Microfluidic Devices Having Isolation Pens and Methods of Testing Biological Micro-Objects with Same

ABSTRACT

A microfluidic device can comprise at least one swept region that is fluidically connected to unswept regions. The fluidic connections between the swept region and the unswept regions can enable diffusion but substantially no flow of media between the swept region and the unswept regions. The capability of biological micro-objects to produce an analyte of interest can be assayed in such a microfluidic device. Biological micro-objects in sample material loaded into a microfluidic device can be selected for particular characteristics and disposed into unswept regions. The sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Flows of medium in the swept region do not substantially affect the biological micro-objects in the unswept regions, but any analyte of interest produced by a biological micro-object can diffuse from an unswept region into the swept region, where the analyte can react with the assay material to produce a localized detectable reaction. Any such detected reactions can be analyzed to determine which, if any, of the biological micro-objects are producers of the analyte of interest.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a non-provisional of (and thus claims the benefit ofand/or priority to) U.S. converted provisional patent application Ser.No. 14/060,321. (The foregoing application Ser. No. 14/060,321 was filedon Oct. 22, 2013 as a regular patent application but converted to aprovisional patent application in response to a Petition filed Oct. 7,2014, which was granted Oct. 9, 2014.) This application is also anon-provisional of (and thus claims the benefit of and/or priority to)U.S. provisional patent application Ser. No. 62/058,658 (filed Oct. 1,2014).

BACKGROUND

As the field of microfluidics continues to progress, microfluidicdevices have become convenient platforms for processing and manipulatingmicro-objects such as biological cells. Some embodiments of the presentinvention are directed to improvements in microfluidic devices andmethods of operating microfluidic devices.

SUMMARY

In some embodiments of the invention, a microfluidic device can includea flow region and a microfluidic sequestration pen. The flow region canbe configured to contain a flow of a first fluidic medium. Themicrofluidic sequestration pen can include an isolation structure and aconnection region. The isolation structure can comprise an isolationregion configured to contain a second fluidic medium. The connectionregion can fluidically connect the isolation region to the flow regionso that, while the flow region and the microfluidic sequestration penare substantially filled with fluidic media: components of the secondmedium are able to diffuse into the first medium or components of thefirst medium are able to diffuse into the second medium; and there issubstantially no flow of the first medium from the flow region into theisolation region.

Some embodiments of the invention include a process of analyzing abiological micro-object in a microfluidic device, which can comprise amicrofluidic channel to which at least one microfluidic sequestrationpen is fluidically connected. The at least one sequestration pen cancomprise a fluidic isolation structure comprising an isolation regionand a connection region fluidically connecting the isolation region tothe channel. The process can include loading one or more biologicalmicro-objects into the at least one sequestration pen, and incubatingthe loaded biological micro-objects for a period of time sufficient toallow the biological micro-objects to produce an analyte of interest.The process can also include disposing capture micro-objects in thechannel adjacent to an opening from the connection region of the atleast one sequestration pen to the channel, and monitoring binding ofthe capture micro-objects to the analyte of interest. The capturemicro-objects can comprise at least one type of affinity agent capableof specifically binding the analyte of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an example of a process in which at least two tests can beperformed on micro-objects in a microfluidic device according to someembodiments of the invention.

FIG. 2A is a perspective view of a microfluidic device with which theprocess of FIG. 1 can be performed according to some embodiments of theinvention.

FIG. 2B is a side, cross-sectional view of the microfluidic device ofFIG. 2A.

FIG. 2C is a top, cross-sectional view of the microfluidic device ofFIG. 2A.

FIG. 3A is a partial side, cross-sectional view of the microfluidicdevice of FIGS. 2A-2C absent the barriers (for ease of illustration) inwhich the selector is configured as a dielectrophoresis (DEP) deviceaccording to some embodiments of the invention.

FIG. 3B is a partial top, cross-section view of FIG. 3A.

FIG. 4A is a perspective view of another example of a microfluidicdevice according to some embodiments of the invention.

FIG. 4B is a side, cross-sectional view of the microfluidic device ofFIG. 4A.

FIG. 4C is a top, cross-sectional view of the microfluidic device ofFIG. 4A.

FIG. 5 illustrates an example of a sequestration pen in which a lengthof a connection region from a channel to an isolation region is greaterthan a penetration depth of medium flowing in the channel according tosome embodiments of the invention.

FIG. 6 is another example of a sequestration pen comprising a connectionregion from a channel to an isolation region that is longer than apenetration depth of medium flowing in the channel according to someembodiments of the invention.

FIGS. 7A-7C show yet another example of a configuration of asequestration pen according to some embodiments of the invention.

FIG. 8 shows an example of loading biological micro-objects into a flowpath of the microfluidic device of FIGS. 2A-2C according to someembodiments of the invention.

FIG. 9 illustrates an example of flowing biological micro-objects into achannel of the microfluidic device of FIGS. 4A-4C according to someembodiments of the invention.

FIG. 10 illustrates an example of testing the biological micro-objectsin the flow path of the microfluidic device of FIGS. 2A-2C for a firstcharacteristic according to some embodiments of the invention.

FIG. 11 is an example of selecting biological micro-objects in themicrofluidic device of FIGS. 2A-2C according to some embodiments of theinvention.

FIG. 12 illustrates an example of selecting biological micro-objects inthe microfluidic device of FIGS. 4A-4C according to some embodiments ofthe invention.

FIG. 13 illustrates an example of moving selected biologicalmicro-objects into holding pens in the microfluidic device of FIGS.2A-2C according to some embodiments of the invention.

FIG. 14 shows an example of flushing biological micro-objects from theflow path of the microfluidic device of FIGS. 2A-2C according to someembodiments of the invention.

FIG. 15 shows an example of moving selected biological micro-objectsfrom the channel into sequestration pens of the microfluidic device ofFIGS. 4A-4C according to some embodiments of the invention.

FIG. 16 is an example of flushing biological micro-objects from achannel in the microfluidic device of FIGS. 4A-4C according to someembodiments of the invention.

FIG. 17 is an example of providing an assay material to the biologicalmicro-objects in the holding pens of the microfluidic device of FIGS.2A-2C according to some embodiments of the invention.

FIG. 18 illustrates the assay material diffused into the holding pens ofthe microfluidic device of FIGS. 2A-2C according to some embodiments ofthe invention.

FIG. 19 shows an example of assay material in the channel of themicrofluidic device of FIGS. 4A-4C and biological micro-objects insequestration pens producing an analyte of interest according to someembodiments of the invention.

FIG. 20 illustrates an example of components of the analyte of interestdiffusing out of isolation regions of sequestration pens and reactingwith assay material adjacent proximal openings to a channel in themicrofluidic device of FIGS. 4A-4C according to some embodiments of theinvention.

FIG. 21 is an example of an assay material comprising labeled capturemicro-objects in the microfluidic device of FIGS. 4A-4C according tosome embodiments of the invention.

FIG. 22 is an example of an assay material comprising a mixture ofcapture micro-objects and a labeling agent in the microfluidic device ofFIGS. 4A-4C according to some embodiments of the invention.

FIG. 23 illustrates examples of a capture micro-object, a component ofthe labeling agent, and the analyte of interest of FIG. 22 according tosome embodiments of the invention.

FIG. 24 shows an example of a composite capture micro-object comprisingmultiple affinity agents according to some embodiments of the invention.

FIG. 25 is a process that illustrates an example of detecting localizedreactions and identifying sequestration pens containing positivebiological micro-objects in a microfluidic device such as the deviceillustrated in FIGS. 4A-4C according to some embodiments of theinvention.

FIG. 26 illustrates moving negative biological micro-objects fromholding pens into the flow path in the device of FIGS. 2A-2C accordingto some embodiments of the invention.

FIGS. 27 shows flushing the negative biological micro-objects from theflow path in the microfluidic device of FIGS. 2A-2C according to someembodiments of the invention.

FIG. 28 illustrates an example of clearing the channel of assay materialin the microfluidic device of FIGS. 4A-4C according to some embodimentsof the invention.

FIG. 29 is an example of separating negative biological micro-objectsfrom positive biological micro-objects in the microfluidic device ofFIGS. 4A-4C according to some embodiments of the invention.

FIG. 30 shows an example of producing clonal biological micro-objects ina sequestration pen in the microfluidic device of FIGS. 4A-4C accordingto some embodiments of the invention.

FIGS. 31A-C depict a microfluidic device comprising a microchannel and aplurality of sequestration pens that open off of the microchannel. Eachsequestration pen contains a plurality of mouse spenocytes. FIG. 31A isa bright field image of a portion of the microchannel device. FIGS. 31Band 31C are fluorescence images obtained using a Texas Red filter. InFIG. 31B, the image was obtained 5 minutes after the start of theantigen specificity assay described in Example 1. In FIG. 31C, the imagewas obtained 20 minutes after the start of the antigen specificity assaydescribed in Example 1. The white arrows in FIG. 31C point tosequestration pens that generated a positive signal in the assay.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the Figures may show simplified or partial views, and the dimensions ofelements in the Figures may be exaggerated or otherwise not inproportion for clarity. In addition, as the terms “on,” “attached to,”or “coupled to” are used herein, one element (e.g., a material, a layer,a substrate, etc.) can be “on,” “attached to,” or “coupled to” anotherelement regardless of whether the one element is directly on, attached,or coupled to the other element or there are one or more interveningelements between the one element and the other element. Also, directions(e.g., above, below, top, bottom, side, up, down, under, over, upper,lower, horizontal, vertical, “x,” “y,” “z,” etc.), if provided, arerelative and provided solely by way of example and for ease ofillustration and discussion and not by way of limitation. In addition,where reference is made to a list of elements (e.g., elements a, b, c),such reference is intended to include any one of the listed elements byitself, any combination of less than all of the listed elements, and/ora combination of all of the listed elements.

As used herein, “substantially” means sufficient to work for theintended purpose. The term “ones” means more than one.

As used herein, the term “micro-object” can encompass one or more of thefollowing: inanimate micro-objects such as microparticles, microbeads(e.g., polystyrene beads, Luminex™ beads, or the like), magnetic beads,microrods, microwires, quantum dots, and the like; biologicalmicro-objects such as cells (e.g., embryos, oocytes, sperms, cellsdissociated from a tissue, blood cells, hydridomas, cultured cells,cells from a cell line, cancer cells, infected cells, transfected and/ortransformed cells, reporter cells, and the like), liposomes (e.g,synthetic or derived from membrane preparations), lipid nanorafts, andthe like; or a combination of inanimate micro-objects and biologicalmicro-objects (e.g., microbeads attached to cells, liposome-coatedmicro-beads, liposome-coated magnetic beads, or the like). Lipidnanorafts have been described, e.g., in Ritchie et al. (2009)“Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,”Methods Enzymol., 464:211-231.

As used herein, the term “cell” refers to a biological cell, which canbe a plant cell, an animal cell (e.g., a mammalian cell), a bacterialcell, a fungal cell, or the like. An animal cell can be, for example,from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, aprimate, or the like.

A “component” of a fluidic medium is any chemical or biochemicalmolecule present in the medium, including solvent molecules, ions, smallmolecules, antibiotics, nucleotides and nucleosides, nucleic acids,amino acids, peptides, proteins, sugars, carbohydrates, lipids, fattyacids, cholesterol, metabolites, or the like.

As used herein in reference to a fluidic medium, “diffuse” and“diffusion” refer to thermodynamic movement of a component of thefluidic medium down a concentration gradient.

The phrase “flow of a medium” means bulk movement of a fluidic mediumprimarily due to any mechanism other than diffusion. For example, flowof a medium can involve movement of the fluidic medium from one point toanother point due to a pressure differential between the points. Suchflow can include a continuous, pulsed, periodic, random, intermittent,or reciprocating flow of the liquid, or any combination thereof. Whenone fluidic medium flows into another fluidic medium, turbulence andmixing of the media can result.

The phrase “substantially no flow” refers to a rate of flow of a fluidicmedium that is less than the rate of diffusion of components of amaterial (e.g., an analyte of interest) into or within the fluidicmedium. The rate of diffusion of components of such a material candepend on, for example, temperature, the size of the components, and thestrength of interactions between the components and the fluidic medium.

As used herein in reference to different regions within a microfluidicdevice, the phrase “fluidically connected” means that, when thedifferent regions are substantially filled with fluid, such as fluidicmedia, the fluid in each of the regions is connected so as to form asingle body of fluid. This does not mean that the fluids (or fluidicmedia) in the different regions are necessarily identical incomposition. Rather, the fluids in different fluidically connectedregions of a microfluidic device can have different compositions (e.g.,different concentrations of solutes, such as proteins, carbohydrates,ions, or other molecules) which are in flux as solutes move down theirrespective concentration gradients and/or fluids flow through thedevice.

In some embodiments, a microfluidic device can comprise “swept” regionsand “unswept” regions. An unswept region can be fluidically connected toa swept region, provided the fluidic connections are structured toenable diffusion but substantially no flow of media between the sweptregion and the unswept region. The microfluidic device can thus bestructured to substantially isolate an unswept region from a flow ofmedium in a swept region, while enabling substantially only diffusivefluidic communication between the swept region and the unswept region.

The capability of biological micro-objects (e.g., biological cells) toproduce specific biological materials can be assayed in such amicrofluidic device. For example, sample material comprising biologicalmicro-objects to be assayed for production of an analyte of interest canbe loaded into a swept region of the microfluidic device. Ones of thebiological micro-objects can be selected for particular characteristicsand disposed in unswept regions. The remaining sample material can thenbe flowed out of the swept region and an assay material flowed into theswept region. Because the selected biological micro-objects are inunswept regions, the selected biological micro-objects are notsubstantially affected by the flowing out of the remaining samplematerial or the flowing in of the assay material. The selectedbiological micro-objects can be allowed to produce the analyte ofinterest, which can diffuse from the unswept regions into the sweptregion, where the analyte of interest can react with the assay materialto produce localized detectable reactions, each of which can becorrelated to a particular unswept region. Any unswept region associatedwith a detected reaction can be analyzed to determine which, if any, ofthe biological micro-objects in the unswept region are sufficientproducers of the analyte of interest.

FIG. 1 illustrates an example of a process 100 for testing micro-objectsin a microfluidic device according to some embodiments of the invention.FIGS. 2A-2C illustrate an example of a microfluidic device 200 withwhich the process 100 can be performed, and FIGS. 3A and 3B illustratean example of a dielectrophoresis (DEP) device that can be part of themicrofluidic device 200. FIGS. 4A-4C illustrate another example of amicrofluidic device 400 with which the process 100 can also beperformed. Neither the device 200 of FIGS. 2A-2C nor the device 400 ofFIGS. 4A-4C, however, is limited to performing the process 100 ofFIG. 1. Nor is the process 100 limited to being performed on the device200 or 400.

As shown in FIG. 1, the process 100 can load a mixture of micro-objectsinto a flow path in a microfluidic device at step 102. The mixtureloaded at step 102 can comprise micro-objects of different types as wellas debris and other objects. At step 104, the process 100 can test themicro-objects in the flow path for a first characteristic, and at step106, the process 100 can separate micro-objects that test positive forthe first characteristic from micro-objects that do not test positive(e.g., micro-objects that test negative) for the first characteristic.As shown, the process 100 can repeat steps 102-106 any number of times.For example, steps 102-106 can be performed k times, after which kmixtures of micro-objects have been loaded at step 102 and sorted atsteps 104, 106 into an initial group of micro-objects all of whichtested positive for the first characteristic. The number k can be anyinteger that is one or greater. (Hereinafter, biological micro-objectsthat test positive to a test are sometimes referred to as “positive”biological micro-objects, and biological micro-objects that do not testpositive to the test (e.g., test negative to the test) are sometimesreferred to as “negative” biological micro-objects.)

The process 100 can then proceed to step 108, where the process 100 canperform a subsequent test on the initial group of micro-objects. Thesubsequent test performed at step 108 can be different than the firsttest performed at step 104. For example, the subsequent test can testfor a subsequent characteristic that is different than the firstcharacteristic tested at step 104. As another example, the subsequenttest performed at step 108 can test for the same characteristic as step104 (the first characteristic mentioned above), but the subsequent testcan have a different sensitivity, accuracy, precision, or the like. Forexample, the subsequent test performed at step 108 can be more sensitiveto the first characteristic than the first test performed at step 104.Regardless, at step 110, the process 100 can separate the micro-objectsthat test positive to the subsequent test at step 108 from themicro-objects that test negative to the subsequent test.

If the first test of step 104 and the subsequent test of step 108 testfor the same characteristic, after steps 108 and 110, micro-objects thattested positive for that characteristic (the first characteristicreferred to above in the discussion of step 104) in response to twodifferent tests have been separated from the k mixtures of micro-objectsloaded into the microfluidic device at k performances of step 102. Asshown, steps 108 and 110 can be repeated, and at each repetition, theprocess 100 can apply a different subsequent test at step 108 that testsfor the same characteristic. Indeed, steps 108 and 110 can be repeated ntimes after which the process 100 has sorted from the k mixtures ofmicro-objects loaded into the microfluidic device at step 102micro-objects that have tested positive n+1 times for the firstcharacteristic tested at steps 104 and 108. The number n can be anyinteger that is one or greater.

As noted, the process 100 can alternatively test at step 108 for asubsequent characteristic that is different than the firstcharacteristic tested at step 104. In such an embodiment, micro-objectshaving both the first characteristic and the subsequent characteristichave been sorted from the k mixtures of micro-objects loaded into themicrofluidic device at step 102. If steps 108 and 110 are repeated, ateach repetition, the process 100 can test for a different subsequentcharacteristic at step 108. For example, at each performance of step108, the process 100 can test for a subsequent characteristic that isnot only different than the first characteristic but also different thanany preceding subsequent characteristic tested during any previous passthrough steps 108 and 110. At each performance of step 110, the process100 can separate the micro-objects that test positive for the subsequentcharacteristic at step 108.

As noted, steps 108 and 110 can be repeated n times. After performingsteps 108 and 110 n times, the process 100 has sorted from the kmixtures of micro-objects loaded into the microfluidic device at step102 micro-objects that have all n+1 of the characteristics tested atsteps 104 and 108. The number n can be an integer that is one orgreater.

Variations of the process 100 are contemplated. For example, in someembodiments, the repetition of step 108 can sometimes test for a newcharacteristic not tested at step 104 or any previous performance ofstep 108 and other times test for the same characteristic tested at step104 or a previous performance of step 108. As another example, at step106 or any repetition of step 110, the process 100 can separate themicro-objects that tested negative from the micro-objects that testedpositive. As yet another example, the process 100 can repeat step 104multiple times before proceeding to step 106. In such an example, theprocess 100 can test for different characteristics at each repetition ofstep 104 and then separate the micro-objects that tested positive ateach repetition of step 104 from micro-objects that tested negative toat least one repetition of step 104. Likewise, step 108 can be repeatedmultiple times before proceeding to step 110.

Examples of microfluidic devices 200 and 400 are now discussed withrespect to FIGS. 2A-7C. Examples of operation of the process 100 withthe devices 200 and 400 in which the micro-objects include biologicalmicro-objects such as biological cells are then described with respectto FIGS. 8-30.

FIGS. 2A-2C illustrate an example of a microfluidic device 200 withwhich the process 100 can be performed. As shown, the microfluidicdevice 200 can comprise a housing 202, a selector 222, a detector 224, aflow controller 226, and a control module 230.

As shown, the housing 202 can comprise one or more flow regions 240 forholding a liquid medium 244. FIG. 2B illustrates an inner surface 242 ofthe flow region 240 on which the medium 244 can be disposed as even(e.g., flat) and featureless. The inner surface 242, however, canalternatively be uneven (e.g., not flat) and comprise features such aselectric terminals (not shown).

The housing 202 can comprise one or more inlets 208 through which themedium 244 can be input into the flow region 240. An inlet 208 can be,for example, an input port, an opening, a valve, another channel,fluidic connectors, or the like. The housing 202 can also comprise oneor more outlets 210 through which the medium 244 can be removed. Anoutlet 210 can be, for example, an output port, an opening, a valve, achannel, fluidic connectors, or the like. As another example, the outlet210 can comprise a droplet outputting mechanism such as any of theoutputting mechanisms disclosed in U.S. patent application Ser. No.13/856,781 filed Apr. 4, 2013 (attorney docket no. BL1-US). All or partof the housing 202 can be gas permeable to allow gas (e.g., ambient air)to enter and exit the flow region 240.

The housing 202 can also comprise a microfluidic structure 204 disposedon a base (e.g., a substrate) 206. The microfluidic structure 204 cancomprise a flexible material, such as rubber, plastic, an elastomer,silicone (e.g., patternable silicone), polydimethylsioxane (“PDMS”), orthe like, which can be gas permeable. Alternatively, the microfluidicstructure 204 can comprise other materials including rigid materials.The base 206 can comprise one or more substrates. Although illustratedas a single structure, the base 206 can comprise multiple interconnectedstructures such as multiple substrates. The micro-fluidic structure 104can likewise comprise multiple structures, which can be interconnected.For example, the micro-fluidic structure 104 can additionally comprise acover (not shown) made from material that is the same as or differentthan the other material in the structure.

The microfluidic structure 204 and the base 206 can define the flowregion 240. Although one flow region 240 is shown in FIGS. 2A-2C, themicrofluidic structure 204 and the base 206 can define multiple flowregions for the medium 244. The flow region 240 can comprise channels(252 in FIG. 2C) and chambers, which can be interconnected to formmicrofluidic circuits. For enclosures that comprise more than one flowregion 240, each flow region 240 can be associated with one or moreinlets 108 and one or more outlets 110 for respectively inputting andremoving medium 144 from the flow region 240.

As shown FIGS. 2B and 2C, the flow region 240 can comprise one or morechannels 252 for the medium 244. For example, the channel 252 can begenerally from the inlet 208 to the outlet 210. As also shown, holdingpens 256 defining non-flow spaces (or isolation regions) can be disposedin the flow region 240. That is, at least a portion of the interior ofeach holding pen 256 can be a non-flow space into which medium 244 fromthe channel 252 does not directly flow except when an empty flow region240 is initially being filled with the medium 244. For example, eachholding pen 256 can comprise one or more barriers 254 that form apartial enclosure the inside of which can include a non-flow space. Thebarriers 254 that define the holding pens 256 can thus prevent medium244 from flowing directly into the protected interior of any of theholding pens 256 from the channel 252 while the flow region 240 isfilled with medium 244. For example, a barrier 254 of a pen 256 cansubstantially prevent bulk flow of the medium 244 from the channel 252into the non-flow spaces of the pens 256 while the flow region 240 isfilled with medium 244, instead allowing substantially only diffusivemixing of medium from the channel 252 with medium in the non-flow spacein a pen 256. Accordingly, exchange of nutrients and waste between thenon-flow space in a holding pen 256 and the channel 252 can occursubstantially only by diffusion.

The foregoing can be accomplished by orienting a pen 256 such that noopening into the pen 256 faces directly into the flow of medium 244 in achannel 252. For example, if the flow of medium is from the inlet 208 tothe outlet 210 (and thus left to right) in the channel 252 in FIG. 2C,each of the pens 256 substantially impedes direct flow of medium 244from the channel 252 into the pens 256 because the openings of each ofthe pens 256 do not face to the left in FIG. 2C, which would be directlyinto such a flow.

There can be many such holding pens 256 in the flow region 240 disposedin any pattern, and the holding pens 256 can be any of many differentsizes and shapes. Although shown as disposed against side walls of themicrofluidic structure 204 in FIG. 2C, one or more (including all) ofthe pens 256 can be stand alone structures disposed away from a sidewallof the microfluidic structure 204 in the channel 252. As shown in FIG.2C, openings of the holding pens 256 can be disposed adjacent thechannel 252, which can be adjacent to the openings of more than one pen256. Although one channel 252 adjacent fourteen pens 256 are shown,there can be more channel 252, and there can be more or fewer pens 256adjacent any particular channel 252.

The barriers 254 of the pens 256 can comprise any of the types ofmaterials discussed above with respect to the microfluidic structure204. The barriers 254 can comprise the same material as the microfluidicstructure 204 or a different material. The barriers 254 can extend fromthe surface 242 of the base 206 across the entirety of the flow region240 to an upper wall (opposite the surface 242) of the microfluidicstructure 204 as shown in FIG. 2B. Alternatively, one or more of thebarriers 254 can extend only partially across the flow region 240 andthus not extend entirely to the surface 242 or the upper wall of themicrofluidic structure 204.

The selector 222 can be configured to create selectively electrokineticforces on micro-objects (not shown) in the medium 244. For example, theselector 222 can be configured to selectively activate (e.g., turn on)and deactivate (e.g., turn off) electrodes at the inner surface 242 ofthe flow region 240. The electrodes can create forces in the medium 244that attract or repel micro-objects (not shown) in the medium 244, andthe selector 222 can thus select and move one or more micro-objects inthe medium 244. The electrodes can be, for example, dielectrophoresis(DEP) electrodes.

For example, the selector 222 can comprise one or more optical (e.g.,laser) tweezers devices and/or one or more optoelectronic tweezers (OET)devices (e.g., as disclosed in U.S. Pat. No. 7,612,355 (which isincorporated in its entirety by reference herein) or U.S. patentapplication Ser. No. 14/051,004 (attorney docket no. BL9-US) (which isalso incorporated in its entirety by reference herein). As yet anotherexample, the selector 222 can include one or more devices (not shown)for moving a droplet of the medium 244 in which one or more ofmicro-objects are suspended. Such devices (not shown) can includeelectrowetting devices such as optoelectronic wetting (OEW) devices(e.g., as disclosed in U.S. Pat. No. 6,958,132). The selector 222 canthus be characterized as a DEP device in some embodiments.

FIGS. 3A and 3B illustrate an example in which the selector 222comprises a DEP device 300. As shown, the DEP device 300 can comprise afirst electrode 304, a second electrode 310, an electrode activationsubstrate 308, a power source 312 (e.g., an alternating current (AC)power source), and a light source 320. Medium 244 in the flow region 240and the electrode activation substrate 308 can separate the electrodes304, 310. Changing patterns of light 322 from the light source 320 canselectively activate and deactivate changing patterns of DEP electrodesat regions 314 of the inner surface 242 of the flow region 240.(Hereinafter the regions 314 are referred to as “electrode regions.”)

In the example illustrated in FIG. 3B, a light pattern 322′ directedonto the inner surface 242 illuminates the cross-hatched electroderegions 314 a in the square pattern shown. The other electrode regions314 are not illuminated and are hereinafter referred to as “dark”electrode regions 314. The relative electrical impedance across theelectrode activation substrate 308 from each dark electrode region 314to the second electrode 310 is greater than the relative impedance fromthe first electrode 304 across the medium 244 in the flow region 240 tothe dark electrode region 314. Illuminating an electrode region 314 a,however, reduces the relative impedance across the electrode activationsubstrate 308 from the illuminated electrode region 314 a to the secondelectrode 310 to less than the relative impedance from the firstelectrode 304 across the medium 244 in the flow region 240 to theilluminated electrode region 314 a.

With the power source 312 activated, the foregoing creates an electricfield gradient in the medium 244 between illuminated electrode regions314 a and adjacent dark electrode regions 314, which in turn createslocal DEP forces that attract or repel nearby micro-objects (not shown)in the medium 244. DEP electrodes that attract or repel micro-objects inthe medium 244 can thus be selectively activated and deactivated at manydifferent such electrode regions 314 at the inner surface 242 of theflow region 240 by changing light patterns 322 projected form a lightsource 320 (e.g., a laser source or other type of light source) into themicrofluidic device 200. Whether the DEP forces attract or repel nearbymicro-objects can depend on such parameters as the frequency of thepower source 312 and the dielectric properties of the medium 244 and/ormicro-objects (not shown).

The square pattern 322′ of illuminated electrode regions 314 aillustrated in FIG. 3B is an example only. Any pattern of the electroderegions 314 can be illuminated by the pattern of light 322 projectedinto the device 200, and the pattern of illuminated electrode regions322′ can be repeatedly changed by changing the light pattern 322.

In some embodiments, the electrode activation substrate 308 can be aphotoconductive material, and the inner surface 242 can be featureless.In such embodiments, the DEP electrodes 314 can be created anywhere andin any pattern on the inner surface 242 of the flow region 240 inaccordance with the light pattern 322 (see FIG. 3A). The number andpattern of the electrode regions 314 are thus not fixed but correspondto the light pattern 322. Examples are illustrated in the aforementionedU.S. Pat. No. 7,612,355, in which the un-doped amorphous siliconmaterial 24 shown in the drawings of the foregoing patent can be anexample of photoconductive material that can compose the electrodeactivation substrate 308.

In other embodiments, the electrode activation substrate 308 cancomprise a circuit substrate such as a semiconductor material comprisinga plurality of doped layers, electrically insulating layers, andelectrically conductive layers that form semiconductor integratedcircuits such as is known in semiconductor fields. In such embodiments,electric circuit elements can form electrical connections between theelectrode regions 314 at the inner surface 242 of the flow region 240and the second electrode 310 that can be selectively activated anddeactivated by the light pattern 322. When not activated, eachelectrical connection can have high impedance such that the relativeimpedance from a corresponding electrode region 214 to the secondelectrode 210 is greater than the relative impedance from the firstelectrode 204 through the medium 144 to the corresponding electroderegion 214. When activated by light in the light pattern 222, however,each electrical connection can have low impedance such that the relativeimpedance from a corresponding electrode region 214 to the secondelectrode 210 is less than the relative impedance from the firstelectrode 204 through the medium 144 to the corresponding electroderegion 214, which activates a DEP electrode at the correspondingelectrode region 214 as discussed above. DEP electrodes that attract orrepel micro-objects (not shown) in the medium 144 can thus beselectively activated and deactivated at many different electroderegions 214 at the inner surface 142 of the flow region 140 by the lightpattern 222. Non-limiting examples of such configurations of theelectrode activation substrate 308 include the phototransistor-baseddevice 300 illustrated in FIGS. 21 and 22 of U.S. Pat. No. 7,956,339 andthe devices 200, 400, 500, and 600 illustrated throughout the drawingsin the aforementioned U.S. patent application Ser. No. 14/051,004.

In some embodiments, the first electrode 304 can be part of a first wall302 (or cover) of the housing 202, and the electrode activationsubstrate 308 and second electrode 310 can be part of a second wall 306(or base) of the housing 202, generally as illustrated in FIG. 3A. Asshown, the flow region 240 can be between the first wall 302 and thesecond wall 306. The foregoing, however, is but an example. In otherembodiments, the first electrode 304 can be part of the second wall 306and one or both of the electrode activation substrate 308 and/or thesecond electrode 310 can be part of the first wall 302. As anotherexample, the first electrode 304 can be part of the same wall 302 or 306as the electrode activation substrate 308 and the second electrode 310.For example, the electrode activation substrate 308 can comprise thefirst electrode 304 and/or the second electrode 310. Moreover, the lightsource 320 can alternatively be located below the housing 202.

Configured as the DEP device 300 of FIGS. 3A and 3B, the selector 222can thus select a micro-object (not shown) in the medium 244 in the flowregion 240 by projecting a light pattern 322 into the device 200 toactivate one or more DEP electrodes at electrode regions 314 of theinner surface 242 of the flow region 240 in a pattern that surrounds andcaptures the micro-object. The selector 222 can then move the capturedmicro-object by moving the light pattern 322 relative to the device 200.Alternatively, the device 200 can be moved relative to the light pattern322.

Although the barriers 254 that define the holding pens 256 areillustrated in FIGS. 2B and 2C and discussed above as physical barriers,the barriers 254 can alternatively be virtual barriers comprising DEPforces activated by light in the light pattern 322.

With reference again to FIGS. 2A-2C, the detector 224 can be a mechanismfor detecting events in the flow region 240. For example, the detector224 can comprise a photodetector capable of detecting one or moreradiation characteristics (e.g., due to fluorescence or luminescence) ofa micro-object (not shown) in the medium. Such a detector 224 can beconfigured to detect, for example, that one or more micro-objects (notshown) in the medium 244 are radiating electromagnetic radiation and/orthe approximate wavelength, brightness, intensity, or the like of theradiation. Examples of suitable photodetectors include withoutlimitation photomultiplier tube detectors and avalanche photodetectors.

The detector 224 can alternatively or in addition comprise an imagingdevice for capturing digital images of the flow region 240 includingmicro-objects (not shown) in the medium 244. Examples of suitableimaging devices that the detector 224 can comprise include digitalcameras or photosensors such as charge coupled devices and complementarymetal-oxide-semiconductor imagers. Images can be captured with suchdevices and analyzed (e.g., by the control module 230 and/or a humanoperator).

The flow controller 226 can be configured to control a flow of themedium 244 in the flow region 240. For example, the flow controller 226can control the direction and/or velocity of the flow. Non-limitingexamples of the flow controller 226 include one or more pumps or fluidactuators. In some embodiments, the flow controller 226 can includeadditional elements such as one or more sensors (not shown) for sensing,for example, the velocity of the flow of the medium 244 in the flowregion 240.

The control module 230 can be configured to receive signals from andcontrol the selector 222, the detector 224, and/or the flow controller226. As shown, the control module 230 can comprise a controller 232 anda memory 234. In some embodiments, the controller 232 can be a digitalelectronic controller (e.g., a microprocessor, microcontroller,computer, or the like) configured to operate in accordance with machinereadable instructions (e.g., software, firmware, microcode, or the like)stored as non-transitory signals in the memory 234, which can be adigital electronic, optical, or magnetic memory device. Alternatively,the controller 232 can comprise hardwired digital circuitry and/oranalog circuitry or a combination of a digital electronic controlleroperating in accordance with machine readable instructions and hardwireddigital circuitry and/or analog circuitry. The controller 130 can beconfigured to perform all or any part of the processes 100, 400disclosed herein.

In some embodiments, the pens 256 can be shielded from illumination(e.g., by the detector 224 and/or the selector 222) or can be onlyselectively illuminated for brief periods of time. Biologicalmicro-objects 502 can thus be protected from further illumination orfurther illumination of the biological micro-objects 502 can beminimized after the biological micro-objects 502 are moved into the pens256.

FIGS. 4A-4C illustrate another example of a microfluidic device 400. Asshown, the microfluidic device 400 can enclose a microfluidic circuit432 comprising a plurality of interconnected fluidic circuit elements.In the example illustrated in FIGS. 4A-4C, the microfluidic circuit 432includes a flow region/channel 434 to which sequestration pens 436, 438,440 are fluidically connected. One channel 434 and three sequestrationpens 436, 438, 440 are shown, but there can be more than one channel 434and more or fewer than three sequestration pens 436, 438, 440 connectedwith any particular channel. The channel 434 and sequestration pens 436,438, 440 are examples of fluidic circuit elements. The microfluidiccircuit 432 can also include additional or different fluidic circuitelements such as fluidic chambers, reservoirs, and the like.

Each sequestration pen 436, 438, 440 can comprise an isolation structure446 defining an isolation region 444 and a connection region 442fluidically connecting the isolation region 444 to the channel 434. Theconnection region 442 can comprise a proximal opening 452 to the channel434 and a distal opening 454 to the isolation region 444. The connectionregion 442 can be configured so that a maximum penetration depth of aflow of a fluidic medium (not shown) flowing at a maximum velocity(V_(max)) in the channel 434 does not extend into the isolation region444. A micro-object (not shown) or other material (not shown) disposedin an isolation region 444 of a pen 436, 438, 440 can thus be isolatedfrom and not substantially affected by a flow of medium (not shown) inthe channel 434. The channel 434 can thus be an example of a sweptregion, and the isolation regions of the sequestration pens 436, 438,440 can be examples of unswept regions. Before turning to a moredetailed discussion of the foregoing, a brief description of themicrofluidic device 400 and examples of an associated control system 470is provided.

The microfluidic device 400 can comprise an enclosure 402 enclosing themicrofluidic circuit 432, which can contain one or more fluidic media.Although the device 400 can be physically structured in different ways,in the example shown in FIGS. 4A-4C, the enclosure 402 is depicted ascomprising a support structure 404 (e.g., a base), a microfluidiccircuit structure 412, and a cover 422. The support structure 404,microfluidic circuit structure 412, and the cover 422 can be attached toeach other. For example, the microfluidic circuit structure 412 can bedisposed on the support structure 404, and the cover 422 can be disposedover the microfluidic circuit structure 412. With the support structure404 and the cover 422, the microfluidic circuit structure 412 can definethe microfluidic circuit 432. An inner surface of the microfluidiccircuit 432 is identified in the figures as 406.

The support structure 404 can be at the bottom and the cover 422 at thetop of the device 400 as illustrated in FIGS. 4A and 4B. Alternatively,the support structure 404 and cover 422 can be in other orientations.For example, the support structure 404 can be at the top and the cover422 at the bottom of the device 400. Regardless, there can be one ormore ports 424 each comprising a passage 426 into or out of theenclosure 402. Examples of a passage 426 include a valve, a gate, apass-through hole, or the like. Two ports 424 are shown but the device400 can have only one or more than two.

The microfluidic circuit structure 412 can define circuit elements ofthe microfluidic circuit 432 or circuits in the enclosure 402. In theexample, illustrated in FIGS. 4A-4C, the microfluidic circuit structure412 comprises a frame 414 and a microfluidic circuit material 416.

The support structure 404 can comprise a substrate or a plurality ofinterconnected substrates. For example, the support structure 404 cancomprise one or more interconnected semiconductor substrates, printedcircuit boards, or the like. The frame 414 can partially or completelyenclose the microfluidic circuit material 416. The frame 414 can be, forexample, a relatively rigid structure substantially surrounding themicrofluidic circuit material 416. For example the frame 414 cancomprise a metal material.

The microfluidic circuit material 416 can be patterned with cavities orthe like to define microfluidic circuit elements and interconnections ofthe microfluidic circuit 432. The microfluidic circuit material 416 cancomprise a flexible material, such as rubber, plastic, elastomer,silicone (e.g., patternable silicone), PDMS, or the like, which can begas permeable. Other examples of materials that can compose microfluidiccircuit material 416 include molded glass, an etchable material such assilicon, photo-resist (e.g., SU8), or the like. In some embodiments,such materials—and thus the microfluidic circuit material 416—can berigid and/or substantially impermeable to gas. Regardless, microfluidiccircuit material 416 can be disposed on the support structure 404 andinside the frame 414.

The cover 422 can be an integral part of the frame 414 and/or themicrofluidic circuit material 416. Alternatively, the cover 422 can be astructurally distinct element (as illustrated in FIGS. 4A and 4B). Thecover 422 can comprise the same or different materials than the frame414 and/or the microfluidic circuit material 416. Similarly, the supportstructure 404 can be a separate structure from the frame 414 ormicrofluidic circuit material 416 as illustrated or an integral part ofthe frame 414 or microfluidic circuit material 416. Likewise the frame414 and microfluidic circuit material 416 can be separate structures asshown in FIGS. 4A-4C or integral portions of the same structure. In someembodiments, the cover 422 and/or the support structure 404 can betransparent to light.

FIG. 4A also illustrates simplified block diagram depictions of examplesof a control/monitoring system 470 that can be utilized in conjunctionwith the microfluidic device 400. As shown, the system 470 can comprisea control module 472 and control/monitoring equipment 480. The controlmodule 472 can be configured to control and monitor the device 400directly and/or through the control/monitoring equipment 480.

The control module 472 can comprise a digital controller 474 and adigital memory 476. The controller 474 can be, for example, a digitalprocessor, computer, or the like, and the digital memory 476 can be anon-transitory digital memory for storing data and machine executableinstructions (e.g., software, firmware, microcode, or the like) asnon-transitory data or signals. The controller 474 can be configured tooperate in accordance with such machine executable instructions storedin the memory 476. Alternatively or in addition, the controller 474 cancomprise hardwired digital circuitry and/or analog circuitry. Thecontrol module 472 can thus be configured to perform all or part of anyprocess (e.g., process 100 of FIG. 1 and/or process 2500 of FIG. 25),step of such a process, function, act, or the like discussed herein.

The control/monitoring equipment 480 can comprise any of a number ofdifferent types of devices for controlling or monitoring themicrofluidic device 400 and processes performed with the microfluidicdevice 400. For example, the equipment 480 can include power sources(not shown) for providing power to the microfluidic device 400; fluidicmedia sources (not shown but can comprise a flow controller like 226 ofFIG. 2A) for providing fluidic media to or removing media from themicrofluidic device 400; motive modules (not shown but can comprise aselector like 222 of FIG. 2A) for controlling selection and movement ofmicro-objects (not shown) in the microfluidic circuit 432; image capturemechanisms (not shown but can be like the detector 224 of FIG. 2A) forcapturing images (e.g., of micro-objects) inside the microfluidiccircuit 432; stimulation mechanisms (not shown) for directing energyinto the microfluidic circuit 432 to stimulate reactions; or the like.

As noted, the control/monitoring equipment 480 can comprise motivemodules for selecting and moving micro-objects (not shown) in themicrofluidic circuit 432. A variety of motive mechanisms can beutilized. For example, dielectrophoresis (DEP) mechanisms (e.g., likethe selector 222 of FIG. 2A) can be utilized to select and movemicro-objects (not shown) in the microfluidic circuit. The base 404and/or cover 422 of the microfluidic device 400 can comprise DEPconfigurations for selectively inducing DEP forces on micro-objects (notshown) in a fluidic medium (not shown) in the microfluidic circuit 432to select, capture, and/or move individual micro-objects. Thecontrol/monitoring equipment 480 can include one or more control modulesfor such DEP configurations.

An example of such a DEP configuration of the support structure 404 orthe cover 422 is an optoelectronic tweezers (OET) configuration.Examples of suitable OET configurations of the support structure 404 orcover 422 and associated monitoring and control equipment areillustrated in the following U.S. patent documents each of which isincorporated herein by reference in its entirety: U.S. Pat. Nos.7,612,355; 7,956,339; U.S. Patent Application Publication No.2012/0325665; U.S. Patent Application Publication No. 2014/0124370; U.S.patent application Ser. No. 14/262,140 (pending); and U.S. patentapplication Ser. No. 14/262,200 (pending). Micro-objects (not shown) canthus be individually selected, captured, and moved within themicrofluidic circuit 432 of the microfluidic device 400 utilizing DEPdevices and techniques such as OET.

As noted, the channel 434 and pens 436, 438, 440 can be configured tocontain one or more fluidic media (not shown). In the example shown inFIGS. 4A-4C, ports 424 are connected to the channel 434 and allow afluidic medium (not shown) to be introduced into or removed from themicrofluidic circuit 432. Once the microfluidic circuit 432 contains thefluidic medium (not shown), flows of fluidic media (not shown) can beselectively generated and stopped in the channel 434. For example, asshown, ports 424 can be disposed at different locations (e.g., oppositeends) of the channel 434, and a flow of medium (not shown) can becreated from one port 424 functioning as an inlet to another port 424functioning as an outlet.

As discussed above, each sequestration pen 436, 438, 440 can comprise aconnection region 442 and an isolation region 444. The connection region442 can comprise a proximal opening 452 to the channel 434 and a distalopening 454 to the isolation region 444. The channel 434 and eachsequestration pen 436, 438, 440 can be configured so that the maximumpenetration depth of a flow of medium (not shown) flowing in the channel434 extends into the connection region 442 but not the isolation region444.

FIG. 5 illustrates a detailed view of an example of a sequestration pen436. Pens 438, 440 can be configured similarly. Examples ofmicro-objects 522 in pen 436 are also shown. As is known, a flow 512 offluidic medium 502 in a microfluidic channel 434 past a proximal opening452 of a pen 436 can cause a secondary flow 514 of the medium 502 intoand/or out of the pen. To isolate micro-objects 522 in the isolationregion 444 of a pen 436 from the secondary flow 514, the length L_(con)of the connection region 442 of the sequestration pen 436 from theproximal opening 452 to the distal opening 454 can be greater than amaximum penetration depth D_(p) of the secondary flow 514 into theconnection region 442 when the velocity of the flow 512 in the channel434 is at a maximum (V_(max)). As long as the flow 512 in the channel434 does not exceed the maximum velocity V_(max), the flow 512 andresulting secondary flow 514 can thus be limited to the channel 434 andthe connection region 442 and kept out of the isolation region 444. Theflow 512 in the channel 434 will thus not draw micro-objects 522 out ofthe isolation region 444. Micro-objects 522 in the isolation region 444will thus stay in the isolation region 444 regardless of the flow 512 inthe channel 432.

Moreover, the flow 512 will not move miscellaneous particles (e.g.,microparticles and/or nanoparticles) from the channel 434 into theisolation region 444 of a pen 436, nor will the flow 512 drawmiscellaneous particles from the isolation region 444 into the channel434. Having the length L_(con) of the connection region 442 be greaterthan the maximum penetration depth D_(p) can thus prevent contaminationof one pen 436 with miscellaneous particles from the channel 434 oranother pen 438, 440.

Because the channel 434 and the connection regions 442 of the pens 436,438, 440 can be affected by the flow 512 of medium 502 in the channel434, the channel 434 and connection regions 442 can be deemed swept (orflow) regions of the microfluidic circuit 432. The isolation regions 444of the pens 436, 438, 440, on the other hand, can be deemed unswept (ornon-flow) regions. For example, a first medium 502 (e.g., components(not shown) in the first medium 502) in the channel 434 can mix with asecond medium 504 (e.g., components (not shown) in the second medium504) in the isolation region 444 substantially only by diffusion of thefirst medium 504 from the channel 434 through the connection region 442and into the second medium 504 in the isolation region 444. Similarly,the second medium 504 (e.g., components (not shown) in the second medium504) in the isolation region 444 can mix with the first medium 504(e.g., components (not shown) in the first medium 502) in the channel434 substantially only by diffusion of the second medium 502 from theisolation region 444 through the connection region 442 and into thefirst medium 502 in the channel 434. The first medium 502 can be thesame medium or a different medium than the second medium 504. Moreover,the first medium 502 and the second medium 504 can start out being thesame, then become different (e.g., through conditioning of the secondmedium by one or more biological micro-objects in the isolation region444, or by changing the medium flowing through the channel 434).

The maximum penetration depth D_(p) of the secondary flow 514 caused bythe flow 512 in the channel 434 can depend on a number of parameters.Examples of such parameters include: the shape of the channel 434 (e.g.,the channel can direct medium into the connection region 442, divertmedium away from the connection region 442, or simply flow past theconnection region 442); a width W_(ch) (or cross-sectional area) of thechannel 434 at the proximal opening 452; a width W_(con) (orcross-sectional area) of the connection region 442 at the proximalopening 452; the maximum velocity V_(max) of the flow 512 in the channel434; the viscosity of the first medium 502 and/or the second medium 504,or the like.

In some embodiments, the dimensions of the channel 434 and sequestrationpens 436, 438, 440 can be oriented as follows with respect to the flow512 in the channel 434: the channel width W_(ch) (or cross-sectionalarea of the channel 434) can be substantially perpendicular to the flow512, the width W_(con) (or cross-sectional area) of the connectionregion 442 at the proximal opening 552 can be substantially parallel tothe flow 512, and the length L_(con) of the connection region can besubstantially perpendicular to the flow 512. The foregoing are examplesonly, and the dimensions of the channel 434 and sequestration pens 436,438, 440 can be in other orientations with respect to each other.

In some embodiments, the width W_(ch) of the channel 434 at a proximalopening 452 can be within any of the following ranges: 50-1000 microns,50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns,70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns,100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, and100-120 microns. The foregoing are examples only, and the width W_(ch)of the channel 434 can be in other ranges (e.g., a range defined by anyof the endpoints listed above).

In some embodiments, the height H_(ch) of the channel 134 at a proximalopening 152 can be within any of the following ranges: 20-100 microns,20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns,30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoingare examples only, and the height H_(ch) of the channel 434 can be inother ranges (e.g., a range defined by any of the endpoints listedabove).

In some embodiments, a cross-sectional area of the channel 434 at aproximal opening 452 can be within any of the following ranges:500-50,000 square microns, 500-40,000 square microns, 500-30,000 squaremicrons, 500-25,000 square microns, 500-20,000 square microns,500-15,000 square microns, 500-10,000 square microns, 500-7,500 squaremicrons, 500-5,000 square microns, 1,000-25,000 square microns,1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000square microns, 1,000-7,500 square microns, 1,000-5,000 square microns,2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000square microns, 2,000-7,500 square microns, 2,000-6,000 square microns,3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000square microns, 3,000-7,500 square microns, or 3,000 to 6,000 squaremicrons. The foregoing are examples only, and the cross-sectional areaof the channel 434 at a proximal opening 452 can be in other ranges(e.g., a range defined by any of the endpoints listed above).

In some embodiments, the length of the connection region L_(con) can bein any of the following ranges: 1-200 microns, 5-150 microns, 10-100microns, 15-80 microns, 20-60 microns, 20-500 microns, 40-400 microns,60-300 microns, 80-200 microns, and 100-150 microns. The foregoing areexamples only, and length L_(con) of a connection region 442 can be in adifferent ranges than the foregoing examples (e.g., a range defined byany of the endpoints listed above).

In some embodiments, the width W_(con) a connection region 442 at aproximal opening 452 can be in any of the following ranges: 20-500microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns,20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns,30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100microns, 40-80 microns, 40-60 microns, 50-250 microns, 50-200 microns,50-150 microns, 50-100 microns, 50-80 microns, 60-200 microns, 60-150microns, 60-100 microns, 60-80 microns, 70-150 microns, 70-100 microns,and 80-100 microns. The foregoing are examples only, and the widthW_(con) of a connection region 442 at a proximal opening 452 can bedifferent than the foregoing examples (e.g., a range defined by any ofthe endpoints listed above).

In other embodiments, the width W_(con) of a connection region 442 at aproximal opening 452 can be in any of the following ranges: 2-35microns, 2-25 microns, 2-20 microns, 2-15 microns, 2-10 microns, 2-7microns, 2-5 microns, 2-3 microns, 3-25 microns, 3-20 microns, 3-15microns, 3-10 microns, 3-7 microns, 3-5 microns, 3-4 microns, 4-20microns, 4-15 microns, 4-10 microns, 4-7 microns, 4-5 microns, 5-15microns, 5-10 microns, 5-7 microns, 6-15 microns, 6-10 microns, 6-7microns, 7-15 microns, 7-10 microns, 8-15 microns, and 8-10 microns. Theforegoing are examples only, and the width W_(con) of a connectionregion 442 at a proximal opening 452 can be different than the foregoingexamples (e.g., a range defined by any of the endpoints listed above).

In some embodiments, a ratio of the length L_(con) of a connectionregion 442 to a width W_(con) of the connection region 442 at theproximal opening 452 of can be greater than or equal to any of thefollowing ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0,7.0, 8.0, 9.0, 10.0, or more. The foregoing are examples only, and theratio of the length L_(con) of a connection region 442 to a widthW_(con) of the connection region 442 at the proximal opening 452 can bedifferent than the foregoing examples.

As illustrated in FIG. 5, the width W_(con) of the connection region 442can be uniform from the proximal opening 452 to the distal opening 454.The width W_(con) of the connection region 442 at the distal opening 454can thus be in any of the ranges identified above for the width W_(con)of the connection region 442 at the proximal opening 452. Alternatively,the width W_(con) of the connection region 442 at the distal opening 454can be larger (e.g., as shown in FIG. 6)or smaller (e.g., as shown inFIGS. 7A-7C) than the width W_(con) of the connection region 442 at theproximal opening 452.

As also illustrated in FIG. 5, the width of the isolation region 444 atthe distal opening 454 can be substantially the same as the widthW_(con) of the connection region 442 at the proximal opening 452. Thewidth of the isolation region 444 at the distal opening 454 can thus bein any of the ranges identified above for the width W_(con) of theconnection region 442 at the proximal opening 452. Alternatively, thewidth of the isolation region 444 at the distal opening 454 can belarger (e.g., as shown in FIG. 6) or smaller (not shown) than the widthW_(con) of the connection region 442 at the proximal opening 452.

In some embodiments, the maximum velocity V_(max) of a flow 512 in thechannel 434 is the maximum velocity that the channel can maintainwithout causing a structural failure in the microfluidic device in whichthe channel is located. The maximum velocity that a channel can maintaindepends on various factors, including the structural integrity of themicrofluidic device and the cross-sectional area of the channel. Forexemplary microfluidic devices of the present invention, the maximumflow velocity V_(max) in a channel having a cross-sectional area ofaround 3,000 to 4,000 square microns is around 10 μL/sec. Alternatively,the maximum velocity V_(max) of a flow 512 in channel 434 can be set soas to ensure that isolation regions 444 are isolated from the flow 512in channel 434. In particular, based on the width W_(con) of theproximal opening 452 of a connection region 442 of a sequestration pen436, 438, 440, V_(max) can be set so as to ensure that the depth ofpenetration D_(p) of a secondary flow 514 into the connection region isless than L_(con). For example, for a sequestration pen having aconnection region with a proximal opening 452 having a width W_(con) ofabout 30 to 40 microns, V_(max) can be set around 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 μL/sec.

In some embodiments, the sum of the length L_(con) of the connectionregion 442 and a corresponding length of the isolation region 444 of asequestration pen 436, 438, 440 can be sufficiently short for relativelyrapid diffusion of components of a second medium 504 in the isolationregion 444 to a first medium 502 in the channel 434. For example, insome embodiments, the sum of (1) the length L_(con) of the connectionregion 442 and (2) the distance between a biological micro-objectlocated in isolation region 444 of a sequestration pen 436, 438, 440 andthe distal opening 454 of the connection region can be in the followingranges: 40 microns to 300 microns, 50 microns to 550 microns, 60 micronsto 500 microns, 70 microns to 180 microns, 80 microns to 160 microns, 90microns to 140 microns, 100 microns to 120 microns, or any rangeincluding one of the foregoing end points. The rate of diffusion of amolecule (e.g., an analyte of interest, such as an antibody) isdependent on a number of factors, including temperature, viscosity ofthe medium, and the coefficient of diffusion D₀ of the molecule. The D₀for an IgG antibody in aqueous solution at 20° C. is around 4.4×10⁻⁷cm²/sec, while the viscosity of biological micro-object culture mediumis around 9×10-4 m2/sec. Thus, for example, an antibody in biologicalmicro-object culture medium at 20° C. can have a rate of diffusion ofaround 0.5 microns/sec. Accordingly, in some embodiments, a time periodfor diffusion from a biological micro-object located in isolation region444 into the channel 434 can be about 10 minutes or less (e.g., 9, 8, 7,6, 5 minutes, or less). The time period for diffusion can be manipulatedby changing parameters that influence the rate of diffusion. Forexample, the temperature of the media can be increased (e.g., to aphysiological temperature such as 37° C.) or decreased (e.g., to 15° C.,10° C., or 4° C.) thereby increasing or decreasing the rate ofdiffusion, respectively.

The configuration of sequestration pen 436 illustrated in FIG. 5 is butan example, and many variations are possible. For example, the isolationregion 444 is illustrated as sized to contain a plurality ofmicro-objects 522, but the isolation region 444 can be sized to containonly one, two, three, four, five, or similar relatively small numbers ofmicro-objects 522. Accordingly, the volume of an isolation region 444can be, for example, at least 3×10³, 6×10³, 9×10³, 1×10⁴, 2×10⁴, 4×10⁴,8×10⁴, 1×10⁵, 2×10⁵, 4×10⁵, 8×10⁵, 1×10⁶, 2×10⁶ cubic microns, or more.

As another example, the sequestration pen 436 is shown extendinggenerally perpendicularly from the channel 434 and thus forminggenerally 90° angles with the channel 434. The sequestration pen 436 canalternatively extend from the channel 434 at other angles such as, forexample, any angle between 30° and 150°.

As yet another example, the connection region 442 and the isolationregion 444 are illustrated in FIG. 5 as substantially rectangular, butone or both of the connection region 442 and the isolation region 444can be other shapes. Examples of such shapes include oval, triangular,circular, hourglass-shaped, and the like.

As still another example, the connection region 442 and the isolationregion 444 are illustrated in FIG. 5 as having substantially uniformwidths. That is, in FIG. 5, the width W_(con) of the connection region442 is shown as being uniform from the proximal opening 452 to thedistal opening 454; a corresponding width of the isolation region 444 issimilarly uniform; and the width W_(con) of the connection region 442and a corresponding width of the isolation region 444 are shown asequal. Any of the foregoing can be different than shown in FIG. 5. Forexample, a width W_(con) of the connection region 442 can vary from theproximal opening 452 to the distal opening 554 (e.g., in the manner of atrapezoid or an hourglass); a width of the isolation region 444 can vary(e.g., in the manner of a triangle or flask); and a width W_(con) of theconnection region 442 can be different than a corresponding width of theisolation region 444.

FIG. 6 illustrates an example of a sequestration pen illustratingexamples of some of the foregoing variations. The pen shown in FIG. 6can replace any of pens 436, 438, 440 in any of the figures ordiscussions herein.

The sequestration pen of FIG. 6 can comprise a connection region 642 andan isolation structure 646 comprising an isolation region 644. Theconnection region 642 can comprise a proximal opening 652 to the channel434 and a distal opening 654 to the isolation region 644. In the exampleillustrated in FIG. 6, the connection region 642 expands such that itswidth W_(con) increases from the proximal opening 652 to the distalopening 654. Other than shape, however, the connection region 642,isolation structure 646, and isolation region 644 can be generally thesame as the connection region 442, isolation structure 446, andisolation region 444 of FIG. 5 as discussed above.

For example, the channel 434 and the sequestration pen of FIG. 6 can beconfigured so that the maximum penetration depth D_(p) of the secondaryflow 514 extends into the connection region 642 but not into theisolation region 644. The length L_(con) of the connection region 642can thus be greater than the maximum penetration depth D_(p), generallyas discussed above with respect to FIG. 5. Also as discussed above,micro-objects 522 in the isolation region 644 will thus stay in theisolation region 644 as long as the velocity of the flow 512 in thechannel 434 does not exceed the maximum flow velocity V_(max). Thechannel 434 and connection region 642 are thus examples of swept (orflow) regions, and the isolation region 644 is an example of an unswept(or non-flow) region.

FIGS. 7A-7C show examples of variations of the microfluidic circuit 432and channel 434 of FIGS. 4A-4C, as well as additional examples ofvariations of sequestration pens 436, 438, 440. The sequestration pens736 shown in FIGS. 7A-7C can replace any of the pens 436, 438, 440 inany of the figures or discussions herein. Likewise, the microfluidicdevice 700 can replace the microfluidic device 400 in any of the figuresor discussions herein.

The microfluidic device 700 of FIGS. 7A-7C can comprise a supportstructure (not visible but can be like 404 of FIG. 4A-4C), amicrofluidic circuit structure 712, and a cover (not visible but can belike 422). The microfluidic circuit structure 712 can comprise a frame714 and microfluidic circuit material 716, which can be the same as orgenerally similar to the frame 414 and microfluidic circuit material 416of FIGS. 4A-4C. As shown in FIG. 7A, the microfluidic circuit 732defined by the microfluidic circuit material 716 can comprise multiplechannels 734 (two are shown but there can be more) to which multiplesequestration pens 736 are fluidically connected.

Each sequestration pen 736 can comprise an isolation structure 746, anisolation region 744 within the isolation structure 746, and aconnection region 742. From a proximal opening 772 at the channel 734 toa distal opening 774 at the isolation structure 736, the connectionregion 742 can fluidically connect the channel 734 to the isolationregion 744. Generally in accordance with the discussion above of FIG. 5,a flow 782 of a first fluidic medium 702 in a channel 734 can createsecondary flows 784 of the first medium 702 from the channel 734 intoand/or out of the connection regions 742 of pens 736 that are connectedto the channel 734.

As illustrated in FIG. 7B, the connection region 742 can include thearea between the proximal opening 772 to a channel 734 and the distalopening 774 to an isolation structure 746. The length L_(con) of theconnection region 742 can be greater than the maximum penetration depthD_(p) of secondary flow 784, in which case the secondary flow 784 willextend into the connection region 742 without being redirected towardthe isolation region 744 (as shown in FIG. 7A). Alternatively, asillustrated in FIG. 7C, the connection region 742 can have a lengthL_(con) that is less than the maximum penetration depth D_(p), in whichcase the secondary flow 784 will extend through the connection region742 and can be redirected toward the isolation region 744. In thislatter situation, the sum of lengths L_(c1) and L_(c2) of connectionregion 742 can be greater than the maximum penetration depth D_(p). Inthis manner, secondary flow 784 will not extend into isolation region744. Whether length L_(con) of connection region 742 is greater than thepenetration depth D_(p) or the sum of lengths L_(c1) and L_(c2) ofconnection region 742 is greater than the penetration depth D_(p), aflow 782 of a first medium 702 in channel 734 that does not exceed amaximum velocity V_(max) will produce a secondary flow having apenetration depth D_(p), and micro-objects (not shown but can be like522 in FIG. 5) in the isolation region 744 of a pen 736 will not bedrawn out of the isolation region 744 by a flow 782 of first medium 702in a channel 734. Nor will the flow 782 in the channel 734 drawmiscellaneous materials (not shown) from a channel 734 into theisolation region 744 of a pen 736 or from the isolation region 744 intothe channel 734. Diffusion is the only mechanism by which components ina first medium 702 in the channel 734 can move from the channel 734 intoa second medium 704 in an isolation region 744 of a pen 736. Likewise,diffusion is the only mechanism by which components in a second medium704 in an isolation region 744 of a pen 736 can move from the isolationregion 744 to a first medium 702 in the channel 734. The first medium702 can be the same medium as the second medium 704, or the first medium702 can be a different medium than the second medium 704. Alternatively,the first medium 702 and the second medium 704 can start out being thesame, then become different (e.g., through conditioning of the secondmedium by one or more biological micro-objects in the isolation region744, or by changing the medium flowing through the channel 734).

As illustrated in FIG. 7B, the width W_(ch) of a channel 734perpendicular to the direction of a flow 782 (see FIG. 7A) in thechannel 734 can be substantially perpendicular to a width W_(con1) ofthe proximal opening 772 and thus substantially parallel to a widthW_(con2) of the distal opening 774. The width W_(con1) of the proximalopening 772 and the width W_(con2) of the distal opening 774, however,need not be substantially perpendicular to each other. For example, anangle between an axis (not shown) on which the width W_(con1) of theproximal opening 772 is oriented and another axis on which the widthW_(con2) of the distal opening 774 is oriented can be other thanperpendicular and thus other than 90°. Examples of alternatively anglesinclude angles in any of the following ranges: between 30° and 90°,between 45° and 90°, between 60° and 90°, or the like.

With regard to the foregoing discussion about microfluidic deviceshaving a channel and one or more sequestration pens, a fluidic medium(e.g., a first medium and/or a second medium) can be any fluid that iscapable of maintaining a biological micro-object in a substantiallyassayable state. The assayable state will depend on the biologicalmicro-object and the assay being performed. For example, if thebiological micro-object is a biological micro-object that is beingassayed for the secretion of a protein of interest, the biologicalmicro-object would be substantially assayable provided that it is viableand capable of expressing and secreting proteins.

FIGS. 8-30 illustrate examples of the process 100 of FIG. 1 testingbiological micro-objects (e.g., biological cells) in the microfluidicdevice 200 of FIGS. 2A-2C or the microfluidic device 400 of FIGS. 4A-4C.The process 100 is not, however, limited to sorting biologicalmicro-objects or operating on the microfluidic devices 200, 400. Nor arethe microfluidic devices 200, 400 limited to performing the process 100.Moreover, while aspects of the steps of process 100 may be discussed inconnection with device 200 but not device 400, or vice versa, suchaspects can be applied in the other device or any other similarmicro-fluidic devices.

At step 102, the process 100 can load biological micro-objects into amicro-fluidic device. FIG. 8 illustrates an example in which biologicalmicro-objects 802 (e.g., biological cells) are loaded into a flow region240 (e.g., the channel 252) of the microfluidic device 200. FIG. 9 showsan example in which sample material 902 comprising biologicalmicro-objects 904 is flowed into a channel 434 of the microfluidicdevice 400.

As shown in FIG. 8 (which like FIGS. 10, 11, 13, 14, 17, 18, 26, and 27,illustrates a partial, top, cross-section view into the flow region 240of the device 200), a mixture of biological micro-objects 802 can beloaded into the channel 252 of the microfluidic device 200. For example,the biological micro-objects 802 can be input into the device 200through the inlet 208 (see FIGS. 2A-2C), and the biologicalmicro-objects 802 can move with a flow 804 of medium 244 in the channel252. The flow 804 can be a convection flow. Once the biologicalmicro-objects 802 are in the channel 252 and adjacent the pens 256, theflow 804 can be stopped or slowed to keep the biological micro-objects802 in the flow channel 252 adjacent to the pens 256 for a timesufficient to perform steps 104 and 106. The mixture of biologicalmicro-objects 802 loaded in the channel 252 can comprise different typesof biological micro-objects and other components such as debris,proteins, contamination, particles, and the like.

FIG. 9 illustrates an example in which sample material 902 comprisingbiological micro-objects 904 is flowed into a channel 434 of themicrofluidic device 400. In addition to the biological micro-objects904, the sample material 902 can comprise other micro-objects (notshown) or materials (not shown). In some embodiments, the channel 434can have a cross-sectional area disclosed herein, e.g., about 3,000 to6,000 square microns or about 2,500 to 4,000 square microns. The samplematerial 902 can be flowed into the channel 434 at a rate disclosedherein, e.g., about 0.05 to 0.25 μL/sec (e.g., about 0.1 to 0.2 μL/secor about 0.14 to 0.15 μL/sec). In some embodiments, the control module472 of FIG. 4A can cause the control/monitoring equipment 480 to flow afirst fluidic medium (not shown) containing the sample material 902through a port 424 into the channel 434. Once the sample material 902 isin the channel 434, flow of the medium (not shown) in the channel 434can be slowed or substantially stopped. Starting and stopping flow ofmedium (not shown) in the channel 434 can include opening and closingvalues (not shown) that comprise the passages 426 of the ports 424.

The biological micro-objects 802, 904 can be any biological micro-object802, 904 to be assayed for production of a particular analyte oranalytes of interest. Examples of biological micro-objects 802, 904include biological micro-objects such as mammalian biologicalmicro-objects, human biological micro-objects, immunological biologicalmicro-objects (e.g., T biological micro-objects, B biologicalmicro-objects, macrophages, etc.), B biological micro-object hydrodomas,stem biological micro-objects (e.g., bone marrow-derived stem biologicalmicro-objects, adipose-derived stem biological micro-objects, etc.),transformed biological micro-objects lines (e.g., transformed CHObiological micro-objects, HeLa biological micro-objects, HEK biologicalmicro-objects, etc.), insect biological micro-objects (e.g., Sf9, Sf21,HighFive, etc.), protozoan biological micro-objects (e.g., Leishmaniatarentolae), yeast biological micro-objects (e.g., S. saccharomyces, P.pastoris, etc.), bacterial biological micro-objects (e.g., E. coli, B.subtilis, B. thuringiensis, etc.), any combination of the foregoing, orthe like. Examples of biological micro-objects 904 also include embryos,such as mammalian embryos (e.g., human, primate, ursidae, canine,feline, bovine, ovis, capra, equus, porcine, etc.), or the like.Examples of the analyte of interest include a protein, a carbohydrate, alipid, a nucleic acid, a metabolite, or the like. Other examples of theanalyte of interest include a material that comprises an antibody suchas an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 sub-class), an IgM, IgA,IgD, or IgE class antibody.

At step 104, the process 100 can perform a first test on the biologicalmicro-objects loaded into the micro-fluidic device at step 102. Step 104can include selecting ones of the biological micro-objects in accordancewith the first test. Alternatively, step 104 can include selecting oneof the biological micro-objects without performing the first test. FIG.10 illustrates an example of a first test performed on the biologicalmicro-objects 802 in the channel 252 of the microfluidic device 200,andFIG. 11 illustrates an example of selecting the biological micro-objects802 in accordance with the first test. (The selected biologicalmicro-objects are labeled 1002 in FIG. 11 and thereafter.) FIG. 12 showsan example in which biological micro-objects 1202, 1204, 1206 areselected from among the micro-objects 904 in the channel 434 of themicrofluidic device 400.

The first test can comprise any number of possible tests. For example,the first test, whether performed in the microfluidic device 200 or 400,can test for a first characteristic of the biological micro-objects 802or biological micro-objects 904. The first test performed at step 104can be any test that tests for any desired characteristic. For example,the desired characteristic can relate to the size, shape, and/ormorphology of the biological micro-objects 802 or biologicalmicro-objects 904. The first test can comprise capturing images of thebiological micro-objects 802 or biological micro-objects 904 andanalyzing the images to determine which of the biological micro-objects802 or biological micro-objects 904 have the desired characteristic. Asanother example, the first test performed at step 104 can determinewhich of the biological micro-objects 802 or biological micro-objects904 exhibit a particular detectable condition that indicates the firstcharacteristic. For example, the first characteristic could beexpression of one or more cell-surface markers and the first testperformed at step 104 could detect the presence or absence of suchcell-surface markers on the biological micro-objects 802, 904. Bytesting for an appropriate cell-surface marker or combination ofcell-surface markers, particular cell types can be identified andselected at step 104. Examples of such particular cell types can includehealthy cells, cancer cells, infected cells (e.g., infected with a virusor a parasite), immunological cells (e.g., B cells, T cells,macrophages), stem cells, and the like.

In the example shown in FIG. 10, the detectable condition of thebiological micro-objects 802 in microfluidic device 200 is radiation ofenergy 1006, which can be, for example, electromagnetic radiation. Thebiological micro-objects 802 can be pre-treated (prior to being loadedinto the microfluidic device 200 or in the channel 252) with an assaymaterial (not shown) that causes the biological micro-objects 802 thathave the first characteristic to radiate energy 1006.

Examples of the first characteristic tested at step 104 can include,without limitation, a biological state (e.g., cell type) or a particularbiological activity of the biological micro-objects 802. For example,the first characteristic can be an observable physical characteristic,such as size, shape, color, texture, surface morphology, identifiablesub-components, or other characteristic marks. Alternatively, the firstcharacteristic can be an assayable characteristic, such as permeability,conductivity, capacitance, response to changes in the environment, orproducing (e.g., expressing, secreting or the like) a particularbiological material of interest. The particular biological material ofinterest can be a cell-surface marker (e.g., a membrane associatedprotein, glycoprotein, or the like). Another example of a particularbiological material of interest is a therapeutic protein, such as anantibody (e.g., IgG-type antibody) that specifically binds to an antigenof interest. Thus, selected biological micro-objects 1002 can be one ormore of the biological micro-objects 802 that test positive forproducing (e.g., expressing) a particular biological material such as acell-surface marker, and unselected biological micro-objects 1004 can bebiological micro-objects 802 that do not test positive for theforegoing. Suitable assay materials with which the biologicalmicro-objects 802 can be pretreated include a reagent that both binds tothe particular biological material of interest and includes labels thatradiate the energy 1206.

As shown in FIG. 11, biological micro-objects 1002 can be selected bytrapping the micro-objects 1002 with a light trap 1102. The light traps1102 can be generated, moved, and turned off in the channel 252 of themicrofluidic device 200 by directing changing patterns of light into thechannel 252 generally as discussed above with respect to FIGS. 3A and3B. Unselected biological micro-objects are labeled 1004 in FIG. 11. Inthe example illustrated in FIG. 11, light traps 1102 are not generatedfor the unselected biological micro-objects 1004.

FIG. 12 illustrates selecting, at step 104, biological micro-objects1202, 1204, 1206 from among the biological micro-objects 904 in thechannel 434 of the microfluidic device 400. The selection can be inresponse to the results of a first test performed at step 104.Alternatively, the selection of micro-objects 1202, 1204, 1206 can be arandom selection and thus made without performing the first test. Ifbased on a first test, step 104 can, for example, comprise selecting thebiological micro-objects 1202, 1204, 1206 for one or more observablephysical characteristics or assayable characteristics, as discussedabove. For example, biological micro-objects 1202, 1204, 1206 can beselected from the micro-objects 904 in the sample material 902 based onany of a number of possible detectable characteristics, such asbiological micro-object-type specific characteristics and/orcharacteristics associate with biological micro-object viability orhealth. Examples of such characteristics include size, shape, color,texture, permeability, conductivity, capacitance, expression ofbiological micro-object-type specific markers, response to changes inthe environment, or the like. In one particular embodiment, biologicalmicro-objects 904 having a rounded shape in cross-section with adiameter in any of the following ranges can be selected from the samplematerial 602: 0.5-2.5 microns, 1-5 microns, 2.5-7.5 microns, 5-10microns, 5-15 microns, 5-20 microns, 5-25 microns, 10-15 microns, 10-20microns, 10-25 microns, 10-30 microns, 15-20 microns, 15-25 microns,15-30 microns, 15-35 microns, 20-25 microns, 20-30 microns, 20-35microns, or 20-40 microns. As another example, biological micro-objects604 whose size is between 100 and 500 microns (e.g., between 100 and 200microns, 150 and 300 microns, 200 to 400 microns, or 250 to 500 microns)can be selected from the sample material 902.

Although the example shown in FIG. 12 illustrates selectingmicro-objects 1202, 1204, 1206 in the channel 434, the sample material902 can alternatively be at least partially in the connection region 442of a pen 436, 438, 440. The micro-objects 1202, 1204, 1206 can thus beselected while in the connection regions 142.

In some embodiments, the control module 472 can perform the first testat step 104 by causing the control/monitoring equipment 480 to captureimages of the biological micro-objects 904 in the sample material 902.The control module 472, which can be configured with known imageanalysis algorithms, can analyze the images and identify ones of thebiological micro-objects 904 that have the desired characteristics.Alternatively, a human user can analyze the captured images.

For assaying characteristics of biological micro-objects, a human userand/or the control module 472 can control the assaying. For example,biological micro-objects such as biological micro-objects can be assayedfor permeability, conductivity, or biological micro-object-type specificmarkers (e.g., using antibodies specific to biologicalmicro-object-surface proteins).

At step 106, the process 100 can separate the selected biologicalmicro-objects or biological micro-objects selected as part of step 104.However, if biological micro-objects are selected without performing afirst step at step 104, step 106 can be skipped or can consist of simplyflushing unselected biological micro-objects out of channel 252 (and,optionally, out of flow region 240 as well). FIGS. 13 and 14 illustratean example in which selected biological micro-objects 1002 are moved tothe holding pens 256 in the microfluidic device 200,and unselectedbiological micro-objects 1004 are flushed out of the channel 252. FIGS.15 and 16 show an example in which selected biological micro-objects1202, 1204, 1206 are moved into the isolation regions 444 of pens 426,438, 440 of the microfluidic device 400, after which the unselectedmicro-objects 904 are flushed out of the flow channel 434.

As noted above with respect to FIG. 11, each biological micro-object1002 can be selected with a light trap 1102. For example, the selector222 (see FIGS. 2A-2C) configured as the DEP device 300 of FIGS. 3A and3B can generate light traps 1102 that trap individual selectedbiological micro-objects 1002. As shown in FIG. 13, the DEP device 300can then move the light traps 1102 into the pens 256, which moves thetrapped selected biological micro-objects 1002 into the pens 256. Asillustrated, each selected biological micro-object 1002 can beindividually trapped and moved into a holding pen 256. Alternatively,more than one selected biological micro-object 1002 can be trapped by asingle trap 1102, and/or more than one selected biological micro-object1002 can be moved into any one pen 256. Regardless, two or more of theselected biological micro-objects 1002 can be selected in the channel252 and moved in parallel into the pens 256.

The light traps 1102 can be part of a changing pattern 322 of lightprojected onto an inner surface 242 of the flow region 240 of themicrofluidic device 200 as discussed above with respect to FIGS. 3A and3B. Once a selected biological micro-object 1002 is in a pen 256, thelight trap 1102 corresponding to that biological micro-object 1002 canbe turned off as illustrated in FIG. 14. The detector 224 can captureimages of all or part of the flow region 240, including images of theselected and unselected biological micro-objects 1002, 1004, the channel252, and the pens 256, and those images can facilitate identifying,trapping, and moving individual selected biological micro-objects 1002into specific pens 256. The detector 224 and/or the selector 222 (e.g.,configured as the DEP device of FIGS. 3A and 3B) can thus be one or moreexamples of a separating means for micro-objects that test positive fora first characteristic (e.g., selected biological micro-objects 1002)from micro-objects that test negative for the first characteristic(e.g., unselected biological micro-objects 1004).

As shown in FIG. 14, with the selected biological micro-objects 1002 inthe pens 256, a flow 804 (e.g., a bulk flow) of the medium 244 can flushthe unselected biological micro-objects 1004 out of the channel 252. Asnoted, after loading the biological micro-objects 904 into the channel252 at step 102, the flow 804 of medium 252 can be stopped or slowed. Aspart of step 106, the flow 804 can be resumed or increased to flush theunselected biological micro-objects 1004 out of the channel 252 and, insome examples, out of the microfluidic device 200 (e.g., through theoutlet 210).

The selected biological micro-objects 1202, 1204, 1206 can be moved intothe isolation regions 444 of the sequestration pens 436, 438, 440 of themicrofluidic device 400 in any of a number of possible ways. Forexample, as discussed above, the enclosure 402 of the microfluidicdevice can include DEP configurations, which can be utilized to captureand move particular ones of the biological micro-objects 904 in thesample material 902.

For example, as illustrated in FIG. 15, the control module 472 can map apath 1512, 1514, 1516 from the channel 434 to the isolation region 444of one of the sequestration pens 436, 438, 440 for each of the selectedbiological micro-objects 1202, 1204, 1206. The control module 472 canthen cause an DEP module (not shown) of the control/monitoring equipment480 to generate and direct changing patterns of light into themicrofluidic circuit 432 to capture and move the selected 1202, 1204,1206 biological micro-objects along the paths 1512, 1514, 1516 into theisolation regions 444 of the sequestration pens 436, 438, 440. Thecontrol module 472 can also store in the memory 476 data identifyingeach of the selected biological micro-objects and the particularsequestration pens 436, 438, 440 into which each selected biologicalmicro-object is moved.

Although one selected biological micro-object 1202, 1204, 1206 per pen436, 438, 444 is shown in the example in of FIG. 15, more than onebiological micro-object 1202, 1204, 1206 be moved into a single pen.Examples of numbers of biological micro-objects that can be moved fromthe sample material 902 into a single pen 136, 138, 140 include thefollowing: 1, 2, 3, 4, 5, 1-50, 1-40, 1-30, 1-20, 1-10, 2-50, 2-40,2-30, 2-20, 2-10, 3-50, 3-40, 3-30, 3-20, 3-10, 4-50, 4-40, 4-30, 4-20,4-10, 5-50, 5-40, 5-30, 5-20, and 5-10. The foregoing are examples only,and other numbers of biological micro-objects 904 can be moved from thesample material 902 into a single pen 436, 438, 440.

In some embodiments, at least part of the sample material 902 can beloaded at step 104 into the isolation regions 444 of the pens 436, 438,440. Also as part of step 104, the micro-objects 1202, 1204, 1206 can beselected in the isolation regions 144. In such embodiments, the samplematerial 902 including the unselected micro-objects 904 can be removedfrom the isolation regions 444 at step 106, leaving only the selectedmicro-objects 1202, 1204, 1206 in the isolation regions 444.

As illustrated in FIG. 16, the channel 434 can be cleared of the samplematerial 902 including unselected micro-objects 904 as part of step 106by flushing the channel 434 with a flushing medium (not shown). In FIG.16, the flow of a flushing medium through the channel 134 is labeled1602. The flow 1602 of the flushing medium can be controlled so that thevelocity of the flow 1602 is maintained below the maximum flow velocityV_(max) corresponding to the maximum penetration depth D_(p) asdiscussed above. As also discussed above, this will keep the selectedbiological micro-objects 1202, 1204, 1206 in the isolation regions 444of their respective pens 436, 438, 440 and prevent material from thechannel 434 or one of the pens 436, 438, 440 from contaminating anotherof the pens. In some embodiments, the flushing medium is flowed into achannel 434 having a cross-sectional area disclosed herein, e.g., about3,000 to 6,000 square microns or about 2,500 to 4,000 square microns.The flushing medium can be flowed into the channel at a rate disclosedherein, e.g., about 0.05 to 5.0 μL/sec (e.g., about 0.1 to 2.0, 0.2 to1.5, 0.5 to 1.0 μL/sec, or about 1.0 to 2.0 μL/sec). Clearing thechannel 434 as part of step 106 can comprise flushing the channel 434multiple times.

In some embodiments, the control module 472 can cause thecontrol/monitoring equipment 480 to clear the channel 434. For example,the control module 472 can cause the control/monitoring equipment 480 toflow a flushing medium through a port 424 into the channel 434 and outof another port 424. The control module 472 can keep the velocity of theflow 1602 below the maximum flow velocity V_(max). For example, for achannel 434 having a cross-sectional area of about 3,000 to 6,000 squaremicrons (or about 2,500 to 4,000 square microns), the control module 472can keep the velocity of the flow 1602 below a V_(max) of 5.0 μL/sec(e.g., 4.0, 3.0, or 2.0 μL/sec).

After steps 102-106, the process 100 has sorted a mixture of biologicalmicro-objects (e.g., 802, 904) in a microfluidic device (e.g., 200, 400)into selected biological micro-objects (e.g., 1004, 1202, 1204, 1206)and unselected biological micro-objects (e.g., 1004, 904). The process100 has also placed the selected biological micro-objects in holdingpens (e.g., 256, 436, 438, 440) in the microfluidic device and flushedthe unselected biological micro-objects away. As discussed above, steps102-106 can be repeated and thus performed k times, where k is one (inwhich case steps 102-106 are performed once but not repeated) orgreater. The result can be numerous selected biological micro-objects inholding pens in the microfluidic device.

It is also noted that step 104 can be performed 1 times testing for upto 1 different characteristics before performing step 106, where 1 is apositive integer one or greater. For example, step 104 can test for afirst characteristic of the biological micro-objects, such as size,shape, morphology, texture, visible markers, or the like, after whichstep 104 can be repeated to test for a subsequent characteristic, suchas an assayable characteristic. Thus, the selected biologicalmicro-objects can comprise biological micro-objects from the group(s) ofbiological micro-objects loaded at step 102 that test positive for asmany as l different characteristics.

As noted, moving the selected biological micro-objects from the channel(e.g., 252, 434) into the pens and flushing the unselected biologicalmicro-objects from the channel is but one example of how step 106 can beperformed. Other examples include, moving the unselected biologicalmicro-objects from the channel into the pens and flushing the selectedbiological micro-objects from the channel. For example, the selectedbiological micro-objects can be flushed from the channel and collectedelsewhere in the microfluidic device or delivered to another device (notshown), where the selected biological micro-objects can be furtherprocessed. The unselected biological micro-objects can later be removedfrom the holding pens and discarded.

At step 108, the process 100 can perform a test on the selectedbiological micro-objects or biological micro-objects. This test can be asubsequent test (e.g., a second test) if a first test was performed aspart of step 104. (Hereinafter, the test performed at step 108 isreferred to as a “subsequent test” to distinguish from the “first test”referred to above in discussing step 104.) As noted above, thesubsequent test performed at step 108 can test for the samecharacteristic (i.e., the first characteristic) as the first test ofstep 104 or a different characteristic. As also noted above, if thesubsequent test performed at step 108 is for the first characteristic(and thus the same characteristic tested at step 104), the subsequenttest can nevertheless be different than the first test. For example, thesubsequent test can be more sensitive than the first test to detectionof the first characteristic.

FIGS. 17 and 18 illustrate an example in which the subsequent testperformed at step 108 is performed in the microfluidic device 200 for anassayable characteristic that is different than the first characteristictested at step 104. FIGS. 19-25 illustrate an example in which the testof step 108 is performed in the microfluidic device 400.

As illustrated in FIG. 17, an assay material 1702 can be flowed 804 intothe channel 252 in sufficient quantity to expose the selected biologicalmicro-objects 1002 in the pens 256 to the assay material 1702. Forexample, although the barriers 254 can impede the assay material 1702from flowing directly from the channel 252 into the interior spaces ofthe pens 256, the assay material 1702 can enter the interior portions ofthe pens 256 and thus reach the selected biological micro-objects 1002in the pens by diffusion. The assay material 1702 can comprise amaterial that reacts with the selected biological micro-objects 1002that have the subsequent characteristic to produce a distinct,detectable condition. The assay material 1702 and the resultingdistinct, detectable condition can be different than any assay materialand condition discussed above with respect to the first testing at step104. A washing buffer (not shown) can also be flowed into the channel252 and allowed to diffuse into the pens 256 to wash the selectedbiological micro-objects 1002.

The detectable condition can be radiation of energy having one or morecriteria such as threshold intensity, frequency in a particularfrequency band, or the like. A color of the biological micro-objects1002 is an example of radiating electromagnetic radiation in aparticular frequency band. In the example shown in FIG. 18, selectedbiological micro-objects 1002 that test positive for the subsequentcharacteristic at step 108 continue to be labeled 1002, but thebiological micro-objects that test negative (e.g., do not test positive)for the subsequent characteristic at step 108 are labeled 1802.

An example of the subsequent characteristic tested at step 410 can beviability of the biological micro-objects 1002. For example, thesubsequent characteristic can be whether the biological micro-objects1002 are alive or dead, and the assay material can be a viability dyesuch as 7-aminoactinomycin D. Such a dye can cause biologicalmicro-objects 1002 that are alive to turn a particular color and/or deadbiological micro-objects to turn a different color. The detector 224(see FIGS. 2A-2C) can capture images of the biological micro-objects1002 in the holding pens 256, and the control module 230 can beconfigured to analyze the images to determine which biologicalmicro-objects exhibit the color corresponding to live biologicalmicro-objects 1002 and/or which exhibit the color corresponding to deadbiological micro-objects 1002. Alternatively, a human operator cananalyze the images from the detector 224. The detector 224 and/or thecontrol module 230 so configured can thus be one or more examples of atesting means for testing micro-objects in a liquid medium in a flowpath in a microfluidic device for a particular characteristic (e.g., thefirst characteristic or a subsequent characteristic).

FIG. 19 illustrates an example in which the test performed at step 108is for an analytic of interest 1902 produced by the selected biologicalmicro-objects 1202, 1204, 1206 in isolation pens 236, 238, 240 of themicrofluidic device 400. Components of the analyte of interest 1902 arelabeled 1904 in FIG. 19. The analyte of interest can be, for example,proteins, nucleic acids, carbohydrates, lipids, metabolites, or othermolecules secreted or otherwise released by specific cell types (e.g.,healthy cells, cancer cells, virus- or parasite-infected cells, cellsexhibiting an inflammatory response, or the like). Particular analytesof interest can be, for example, growth factors, cytokines (e.g.,inflammatory or otherwise), viral antigens, parasite antigens, cancercell-specific specific antigens, or therapeutic agents (e.g.,therapeutic agents, such as hormones or therapeutic antibodies).

In the example illustrated in FIG. 19, step 108 can include loading anassay material 1910 into the microfluidic device 400 and detectinglocalized reactions, if any, of analyte components 1904. Step 108 canalso include providing an incubating period after loading the assaymaterial 1910 into the channel 434.

As shown in FIG. 19, the assay material 1910 can substantially fill thechannel 434 or at least areas immediately adjacent the proximal openings442 of the pens 436, 438, 440. Also, the assay material 110 can extendinto the connection regions 442 of at least some of the sequestrationpens 436, 438, 440. In some embodiments, the assay material is flowedinto a channel 434 having a cross-sectional area disclosed herein, e.g.,about 3,000 to 6,000 square microns or about 2,500 to 4,000 squaremicrons. The assay material can be flowed into the channel at a ratedisclosed herein, e.g., about 0.02 to 0.25 μL/sec (e.g., about 0.03 to0.2 μL/sec, or about 0.05 to 0.15 μL/sec, with slower speeds used forbiological cellular assay materials and higher speeds used fornon-cellular assay materials). Once the assay material 1910 is loadedinto place in the channel 434, flow in the channel 434 can be slowed orsubstantially stopped.

The assay material 1910 can be flowed into the channel 434 sufficientlyfast so that the assay material 1910 is in place adjacent the proximalopenings 452 of the pens 436, 438, 440 before analyte components 1904produced in any of the pens 436, 438, 440 can diffuse into the channel434. This can avoid a problem of analyte components 1904 from one pen436, 438, 440 contaminating the channel 434 and/or other pens betweenthe time when selected biological micro-objects 1202, 1204, 1206 aredisposed into the pens 436, 438, 440 and completion of the loading ofthe assay material 1910 into the channel 434.

The velocity at which the assay material 1910 is loaded into the channel434 can thus be at least a minimum flow velocity V_(min) that fullyloads the assay material 1910 into place adjacent the proximal openings452 over a time period T_(load) that is less than a minimum time periodT_(diff) for a substantial amount of analyte components 1904 to diffusefrom an isolation region 444 of a pen 436, 438, 440 into the channel434. A “substantial amount” as used in this context means a detectableamount of analyte components that is sufficient to interfere withaccurate detection of which sequestration pen the analyte componentscame from). The minimum flow velocity V_(min) can be a function of avariety of different parameters. Examples of such parameters include thelength of the channel 434, the length L_(con) of a connection region 442of a pen 436, 438, 440, a diffusion rate of analyte components 1904,medium viscosity, ambient temperature, or the like. Examples of theminimum flow velocity V_(min) include at least about 0.04 μL/sec (e.g.,at least about 0.10, 0.11, 0.12, 0.13, 0.14, μL/sec, or higher).

The minimum flow velocity V_(min) for loading assay material 1910 intothe channel 434 can be less than the maximum flow velocity V_(max)corresponding to a penetration depth D_(p) that is less than the lengthL_(con) of a connection region 442 of a pen 436, 438, 440 as discussedabove. For example, a ratio of V_(max)/V_(min) can be in any of thefollowing ranges: about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,40, 50, 100, or more.

The incubation period provided after loading the assay material 1910 canbe sufficient for the biological micro-objects 1202, 1204, 1206 toproduce the analyte of interest 1902 and for analyte components 1904 todiffuse from the isolation regions 444 of the pens 436, 438, 440 tocorresponding connection regions 442 or proximal openings 452. Forexample, the incubation period can provide analyte components 1904sufficient time to diffuse into the channel 434.

The incubation period can comprise merely passively allowing thebiological micro-objects 1202, 1204, 1206 to naturally produce theanalyte of interest 1902 in the sequestration pens 436, 438, 440.Alternatively, the incubation period can comprise actively stimulatingthe biological micro-objects 1202, 1204, 1206 to produce the analyte ofinterest 1902 by, for example, providing nutrients, growth factors,and/or inductive factors to the biological micro-objects 1202, 1204,1206; controlling the temperature, chemical composition, pH, or the likeof the medium in the isolation regions 444 of the sequestration pens436, 438, 440; directing stimulating energy such as light into theisolation regions 444; or the like.

The term “incubation” and “incubate,” as used herein, cover theforegoing range from merely passively allowing the biologicalmicro-objects 1202, 1204, 1206 to naturally produce analyte 1902 in thesequestration pens 436, 438, 440 to actively stimulating production ofthe analyte. Stimulating the production of analyte 1902 can also includestimulating the growth of a biological micro-object 1202, 1204, 1206.Thus, for example, biological micro-objects 1202, 1204, 1206 can bestimulated to grow prior to and/or while they are being stimulated toproduce an analyte of interest 1902. If the biological micro-objects1202, 1204, 1206 have been loaded into sequestration pens 436, 438, 440as single biological micro-objects, growth stimulation can result in theproduction of clonal biological micro-object populations which expressand/or secrete (or can be stimulated to express and/or secrete) ananalyte of interest.

In some embodiments, the control module 472 can cause thecontrol/monitoring equipment 480 to perform one or more actions duringthe incubation period 150. For example, the control module 472 can causethe control/monitoring equipment 480 to provide growth medium and/orinductive medium either periodically or as a continuous flow.Alternatively, control module 472 can cause the control/monitoringequipment 480 to incubate the biological micro-objects for a period oftime sufficient for the analyte of interest to diffuse into the channel434. For example, in the case of a protein analyte such as an antibody,the control module 472 can provide time for diffusion equal to around 2seconds for every 1 micron that the biological micro-object is separatedfrom the channel 434. For proteins and other analytes significantlysmaller than an antibody, the time needed for diffusion may be smaller,such as 1.5 seconds for every 1 micron, or less (e.g., 1.25 s/μm, 1.0s/μm, 0.75 s/μm, 0.5 s/μm, or less). Conversely, for proteins or otheranalytes significantly larger than an antibody, the time allotted fordiffusion may be larger, such as 2.0 seconds for every micron, or more(e.g., 2.25 s/μm, 2.5 s/μm, 2.75 s/μm, 3.0 s/μm, or more).

It is noted that the incubation period can continue during performanceof subsequent steps of the process 100. Also, the incubation period canbegin prior to completion of step 106 (e.g., during any of steps102-106).

The assay material 1910 can be configured both to interact with analytecomponents 1904 of an analyte of interest 902 and to produce adetectable reaction from the interaction. As illustrated in FIG. 20,analyte components 1904 from biological micro-objects 1202, 1204 insequestration pens 436, 438 interact with the assay material 1910adjacent to the proximal openings 452 of the sequestration pens 436, 438to produce localized, detectable reactions. The biological micro-object1206 in sequestration pen 440 does not, however, produce the analyte ofinterest 1902. Consequently, no such localized reaction (e.g., like2002) occurs adjacent the distal opening 452 of sequestration pen 440.

The localized reactions 2002 can be detectable reactions. For example,the reactions 2002 can be localized luminescence (e.g., fluorescence).Moreover, the localized reactions 2002 can be sufficiently localized andseparated to be separately detectable by a human observer, a camera (notshown) in the control/monitoring equipment 480 of FIG. 4A, or the like.For example, the channel 434 can be sufficiently filled with the assaymaterial 1910 that reactions (e.g., like 2002) are localized, that is,limited to space immediately adjacent the proximal opening 452 of acorresponding sequestration pen 436, 438. As will be seen, the reactions2002 can be from an aggregation of multiple components of the assaymaterial 1910 immediately adjacent one or more of the proximal openings452 of the sequestration pens 436, 438, 440.

Proximal openings 452 of contiguous sequestration pens 436, 438, 440 canbe spaced apart by at least a distance D_(s) (see FIG. 4C) that issufficient to render localized reactions (e.g., like 2002) at adjacentdistal openings 452 distinguishable one from another, for example, by ahuman observer, in images captured by a camera, or the like. Examples ofsuitable distances D_(s) between proximal openings 452 of contiguoussequestration pens 436, 438, 440 include at least 20, 25, 30, 35, 40,45, 50, 55, 60 microns, or more. Alternatively, or in addition,components of the assay material 910 (e.g., capture micro-objects, suchas biological micro-objects, beads, and the like) can be organized infront of sequestration pens. For example, using DEP forces or the like,capture micro-objects can be grouped together and concentrated inregions of the channel 434 located adjacent to the proximal openings 452of sequestration pens 436, 438, 440.

As noted, the assay material 1910, including components such as capturemicro-objects (e.g., biological micro-objects, beads, or the like), canenter and thus be disposed at least in part in the connection regions442 of the sequestration pens 436, 438, 440. In such a case, thereactions 2002, 2004 can occur entirely, substantially entirely, orpartially in the connection regions 442 as opposed to substantiallyentirely in the channel 434. Moreover, capture micro-objects (e.g.,biological micro-objects, beads, or the like) in the assay material 1910can be disposed into isolation regions 444. For example, DEP forces orthe like can be used to select and move capture micro-objects intoisolation regions 444. For capture micro-objects that are disposed inthe isolation region of a sequestration pen, the capture micro-objectscan be disposed proximal to the biological micro-object(s) and/or in aportion (e.g., a sub-compartment) of the isolation region that isdistinct from the portion occupied by the biological micro-object(s).

The assay material 1910 can be any material that specifically interacts,either directly or indirectly, with the analyte of interest 1902 toproduce a detectable reaction (e.g., 2002). FIGS. 19-23 illustrateexamples in which the analyte comprises an antibody with twoantigen-binding sites. As persons skilled in the art will understand,the same examples could be readily adapted for situations in which theanalyte of interest is something other than an antibody withtwo-antigen-binding sites.

FIG. 21, which shows part of the channel 434 and the proximal opening452 of the sequestration pen 436, illustrates an example of the assaymaterial 1910 comprising labeled capture micro-objects 2112. Eachlabeled capture micro-object 2112 can comprise both a binding substancecapable of specifically binding analyte components 1904 and a labelingsubstance. As analyte components 1904 diffuse towards the proximalopening 452 of the sequestration pen 436, labeled capture micro-objects2112 immediately adjacent the opening 452 (or within the sequestrationpen) can bind the analyte components 1904, which can result in alocalized reaction 2002 (e.g., aggregation of the labeled capturemicro-objects 2112) immediately adjacent (or internal to) the proximalopening 452.

Binding of analyte components 1904 to labeled capture micro-objects 2112is greatest when the labeled capture micro-objects 2112 are immediatelyadjacent or internal to a proximal opening 452. This is because theconcentration of analyte components 1904 is highest in isolation region444 and connection region 442, thereby favoring binding of the analytecomponents 1904 to the labeled capture micro-objects 2112 andfacilitating their aggregation in those regions. As analyte components1904 diffuse out into the channel 234 and away from the proximal opening252, their concentration goes down. As a result, fewer analytecomponents 1904 bind to labeled capture micro-objects 2112 that arelocated away from the proximal opening 252. The reduction in binding ofanalyte components 1904 to labeled capture micro-objects 2112 results,in turn, in reduced aggregation of the labeled capture micro-objects2112 located away from the proximal opening 452. Labeled capturemicro-objects 2112 that are not immediately adjacent (or internal) to aproximal opening 452 of a pen 436, 438, 440 thus do not produce adetectable localized reaction 2002 (or produce a localized reaction 2002that is detectably lower in magnitude than the localized reaction 2002that takes place immediately adjacent or internal to the proximalopening 452).

For analyte components that do not have two binding sites for a bindingsubstance on the labeled capture micro-objects 2112, the labeled capturemicro-objects could include two different binding substances (asdiscussed below and shown in FIG. 23), each of which is capable of beingspecifically bound by the analyte components. Alternatively, the assaycould work if the analyte components multimerize (e.g., form homodimers,homotrimers, etc.).

Examples of labeled capture micro-objects 2112 include both inanimateand biological micro-objects. Examples of inanimate micro-objectsinclude micro-structures such as microbeads (e.g., polystyrenemicrobeads), microrods, magnetic beads, quantum dots, and the like. Themicro-structures can be large (e.g., 10-15 microns in diameter, orlarger) or small (e.g., less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1microns in diameter, or smaller). Examples of biological micro-objectsinclude biological micro-objects (e.g., reporter biologicalmicro-objects), liposomes (e.g., synthetic or derived from membranepreparations), microbeads coated with liposomes, lipid nanorafts (see,e.g., Ritchie et al. (2009) “Reconstitution of Membrane Proteins inPhospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231), and thelike.

FIG. 22 illustrates an example of the assay material 1910 comprising amixture of capture micro-objects 2212 and labeling agent, components ofwhich are identified as 2222 and referred to hereinafter as “labels2222”. FIG. 23 shows example configurations of a capture micro-object2212, an analyte component 1904, and a label 2222. The capturemicro-object 2212 can comprise a first affinity agent 2312 thatspecifically binds a first region 2302 of the analyte component 1904.The label 2222 can comprise a second affinity agent 2322 thatspecifically binds a second region 2304 of the analyte component 1904.As illustrated in FIG. 22, a reaction 2002 occurs when the first region2302 of an analyte component 1904 binds to the first affinity agent 2312of a capture micro-object 2212 and the second region 2304 of the analytecomponent 1904 binds to the second affinity agent 2322 of a label 2222.

As analyte components 1904 produced by the biological micro-object 1202in the isolation region 444 of sequestration pen 436 diffuse towards theproximal opening 452, the analyte components 1904 can bind to a capturemicro-object 2212 and a label 2222 immediately adjacent (or internal) toopening 452, thereby resulting in accumulation of label 2112 on thesurface of the capture micro-object 2212. Binding of analyte components1904 to labeled capture micro-objects 2212 is greatest when the capturemicro-objects 2212 are immediately adjacent (or internal) to a proximalopening 452. Similar to the discussion above, this is because therelatively high concentration of analyte components 1904 in isolationregion 444 and connection region 442 facilitate the binding of analytecomponents 1904 to the capture micro-objects 2212 and concomitantassociation of label 2222 at the surface of the capture micro-objects2212. As analyte components 1904 diffuse out into the channel 434 andaway from the proximal opening 452, the concentration goes down andfewer analyte components 1904 bind to capture micro-objects 2212 thatare located away from the proximal opening 452. The reduction in bindingof analyte components 1904 to capture micro-objects 2212 results inreduced accumulation of label 2222 at the surface of the capturemicro-objects 2112 located away from the proximal opening 452.Accordingly, capture micro-objects 2212 that are not immediatelyadjacent (or internal) to a proximal opening 452 of a pen 436, 438, 440do not become detectably labeled or, to the extent that they do becomelabeled, the labeling is detectably lower in magnitude than the labelingthat takes place immediately adjacent or internal to the proximalopening 452.

Examples of capture micro-objects 2212 include all of the examplesidentified above for labeled capture micro-object 2112. Examples of thefirst affinity agent 2312 include a receptor that specificallyrecognizes the analyte components 1904 or a ligand that is specificallyrecognized by the analyte components 1904. For example, in the case ofan antibody analyte, the first affinity agent 2312 can be an antigen ofinterest.

Examples of labels 2222 include labeling agents comprising luminescentlabels (e.g., fluorescent labels) and labeling agents comprising enzymescapable of cleaving a signal molecule that fluoresces upon cleavage.

Examples of the assay material 1910 include assay materials comprisingcomposite capture micro-objects that include multiple affinity agents.FIG. 24 illustrates an example of a composite capture micro-object 2412that comprises a first affinity agent 2402 and a second affinity agent2404. The first affinity agent 2402 can be capable of specificallybinding the first region 2302 of an analyte component 1904 (see FIG.23), and the second affinity agent 2404 can be capable of specificallybinding the second region 2304 of the same analyte component 1904 or adifferent analyte component. Moreover, the first affinity agent 2402 andthe second affinity agent 2404 can optionally bind the first region 2302and second region 2304 of an analyte component 1904 at the same time.

Examples of the first affinity agent 2402 include those discussed above.Examples of the second affinity agent 2404 include a receptor thatspecifically recognizes the second region 2304 of the analyte components1904 or a ligand that is specifically recognized by the second region2304 of the analyte components 1904. For example, in the case of anantibody analyte, the second affinity agent 2404 can bind to theconstant region of an antibody. Examples of the foregoing include an Fcmolecule, an antibody (e.g., an anti-IgG antibody), Protein A, ProteinG, and the like.

Another example of the assay material 1910 is one that comprisesmultiple capture micro-objects. For example, the assay material 1910 cancomprise first capture micro-objects (not shown) comprising the firstaffinity agent 2402 and second capture micro-objects (not shown)comprising the second affinity agent 2404. The first capturemicro-objects can be different than the second capture micro-objects.For example, the first capture micro-objects can have a size, color,shape, or other characteristic that distinguishes the first capturemicro-objects from the second capture micro-objects. Alternatively, thefirst capture micro-objects and the second capture micro-objects can besubstantially the same type of capture micro-objects, with the exceptionof the type of affinity agent each comprises.

Another example of the assay material 1910 is one that comprisesmultiple types of capture micro-objects, each of which is designed tobind to a different analyte of interest. For example, the assay material1910 can comprise first capture micro-objects (not shown) comprising afirst affinity agent and second capture micro-objects (not shown)comprising a second affinity agent, wherein the first and secondaffinity agents do not bind to the same analyte of interest. The firstcapture micro-objects can have a size, color, shape, label, or othercharacteristic that distinguishes the first capture micro-objects fromthe second capture micro-objects. In this manner, multiple analytes ofinterest can be screened for at the same time.

Regardless of the specific content of the assay material 1910, in someembodiments, the control module 472 can cause the control/monitoringequipment 480 to load the assay material 1910 into the channel 434. Thecontrol module 472 can keep the flow of the assay material 1910 in thechannel 434 between the minimum flow velocity V_(min) and the maximumflow velocity V_(max) discussed above. Once the assay material 1910 isin place adjacent the proximal openings 452 of the pens 436, 438, 440,the control module 472 can substantially stop the flow of the assaymaterial 1910 in the channel 434.

Performed in the microfluidic device 400, step 108 can include detectinglocalized reactions 2002 immediately adjacent one or more of theproximal openings 452 of the sequestration pens 436, 438, 440 thatindicate reaction of analyte components 1904 with the assay material1910 loaded into the channel 434. If localized reactions 2002 aredetected immediately adjacent any of the proximal openings 452 of thesequestration pens 436, 438, 440, it can be determined whether any ofthose detected localized reactions 2002 indicate positive performance ofone or more of the biological micro-objects 1202, 1204, 1206 in thesequestration pens 436, 438, 440. In some embodiments, a human user canobserve the channel 434 or connections regions 442 of the pens 436, 438,440 to monitor for and determine whether localized reactions 2002indicate positive performance of biological micro-object 1202, 1204,1206. In other embodiments, the control module 472 can be configured todo so. The process 2500 of FIG. 25 is an example of operation of thecontrol module 472 for performing to monitor for and determine whetherlocalized reactions 2002 indicate positive performance of biologicalmicro-object 1202, 1204, 1206.

At step 2502, the control module 472 performing the process 2500 cancapture at least one image of the channel 434 or connection regions 442of the sequestration pens 436, 438, 440 with a camera or other imagecapture device (not shown but can be an element of thecontrol/monitoring equipment 480 of FIG. 1A). Examples of exposure timesfor capturing each image include 10 ms to 2 seconds, 10 ms to 1.5seconds, 10 ms to 1 second, 50 to 500 ms, 50 to 400 ms, 50 to 300 ms,100 to 500 ms, 100 to 400 ms, 100 to 300 ms, 150 to 500 ms, 150 to 400ms, 150 to 300 ms, 200 to 500 ms, 200 to 400 ms, or 200 to 300ms. Thecontrol module 472 can capture one such image or multiple images. If thecontrol module 472 captures one image, that image can be the final imagereferred to below. If the control module 472 captures multiple images,the control module 472 can combine two or more of the captured imagesinto a final image. For example, the control module 472 can average twoor more of the captured images. In some embodiments, the control module472 can capture and average at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more of thecaptured images to produce the final image.

At step 2504, the control module 472 can identify in the final image anyindications of localized reactions 2002. As discussed above, examples ofthe localized reactions 2002 include luminescence (e.g., fluorescence),and the control module 472 can thus analyze the final image forluminescence immediately adjacent any of the proximal openings 452 ofthe sequestration pens 436, 438, 440. The control module 472 can beprogrammed to utilize any image processing technique to identifylocalized reactions 2002 in the final image. In the example, illustratedin FIG. 20, the control module 472 can detect the localized reactions2002 immediately adjacent the proximal openings 452 of sequestrationpens 436, 438.

At step 2506, the control module 472 can correlate each localizedreaction 2002 detected at step 2504 to a corresponding sequestration pen436, 438, 440. For example, the control module 472 can do so bycorrelating each localized reaction 2002 detected at step 2504 to thesequestration pen 436, 438, 440 with the nearest proximal opening 452 tothe reaction 1002. In the example of FIG. 20, the control module 472 cancorrelate the reactions 2002 to the sequestration pens 436, 438.

The control module 472 can perform steps 2508 and 2510 of FIG. 25 foreach sequestration pen 436, 438, 440 to which a detected reaction wascorrelated at step 2506. With respect to the example of FIG. 20, thecontrol module 472 can thus perform steps 2508 and 2510 forsequestration pen 436 and then repeat steps 2508 and 2510 forsequestration pen 438.

At step 2508, the control module 472 can determine whether the detectedreaction 1002 correlated to the current sequestration pen 436 indicatesa positive result for the biological micro-object(s) 1202 in the currentpen 436. For example, the control module 472 can extract data regardingthe detected reaction 1002 from the final image obtained at step 2502,and determine whether the extracted data indicates a positive result.Any number of different criteria can be used. For example, the detectedreaction 2002 can be luminescence, and the criteria for determining apositive result can include intensity of the luminescence exceeding athreshold, brightness of the luminescence exceeding a threshold, colorof the luminescence falling within a predetermined color range, or thelike. If at step 2508, the control module 472 determines that thedetected reaction is positive, the control module 472 can proceed tostep 2510, where the control module 472 can identify the currentsequestration pen 436 as containing a positive biological micro-object1202. If the determination at step 2508 is negative, the control module472 can repeat step 2508 for the next sequestration pen 438 for which adetected reaction was correlated at step 2506.

In the example illustrated in FIG. 20, it is assumed that the localizedreaction 2002 correlated to sequestration pen 436 is determined at step2508 to be positive, but the localized reaction 2002 correlated tosequestration pen 438 is negative (e.g., luminescence is detected, butit is below the threshold for determining that sequestration pen 438 ispositive). As previously noted, no reaction was detected adjacent theproximal opening 452 of sequestration pen 440. Consequently, the controlmodule 472 identifies only sequestration pen 436 as having a positivebiological micro-object. Although not shown in FIG. 25, the controlmodule 472 can, as part of process 2500, identify sequestration pens438, 440 as negative.

Returning to FIG. 1, at step 110, the process 100 can separate thebiological micro-objects that tested positive at step 108 from thebiological micro-objects that tested negative. FIGS. 26 and 27illustrate an example in which the biological micro-objects 1002 thattested negative for the subsequent characteristic at step 108 are movedinto and then flushed out of the channel 252 of the microfluidic device200. FIG. 29 shows an example in which negative biological micro-objects1204, 1206 are separated from positive biological micro-object 1202 inthe microfluidic device 400.

As shown in FIG. 26, each biological micro-object 1002 that testednegative at step 110 can be selected and trapped with a light trap 2602in a holding pen 256. Negative micro-objects are labeled 1802 in FIG.26. The light trap 2602 can then be moved from a holding pen 256 intothe channel 252. As shown in FIG. 27, the traps 2602 can be turned offin the channel 252, and a flow 804 (e.g., a convection flow) of medium244 can flush the negative biological micro-objects 1802 out of thechannel 252 (and, optionally, out of the flow region 240). The assaymaterial 1702 can diffuse out of the pens 256, and the flow 804 can alsoflush the assay material 1702 out of the channel 252.

The light traps 2602 can be generated and manipulated as discussedabove. For example, as illustrated, each negative biologicalmicro-object 2602 can be individually trapped and moved from a holdingpen 256 into the channel 252. Alternatively, more than one negativebiological micro-object 2602 can be trapped by a single trap 2602. Forexample, there can be more than one biological micro-object 2602 in asingle pen 256. Regardless, two or more of the negative biologicalmicro-objects 2602 can be selected in the pens 256 and moved in parallelinto the channel 252.

The detector 224 can capture images of all or part of the flow region240 including images of the biological micro-objects 1002 in the pens256, and those images can facilitate identifying, trapping, and movingindividual negative biological micro-objects 2602 out of specific pens256 and into the channel 252. The detector 224 and/or the selector 222(e.g., configured as the DEP device of FIGS. 3A and 3B) can thus be oneor more examples of a separating means for micro-objects that testpositive for a characteristic from micro-objects that test negative forthe characteristic.

As shown in FIG. 27, with the negative biological micro-objects 1802 inthe channel 252, a flow 804 of the medium 244 can flush the biologicalmicro-objects 1802 out of the channel 252 and, in some examples, out ofthe microfluidic device 200 (e.g., through the outlet 210). For example,if the flow 804 was previously stopped or slowed, the flow 804 can beresumed or increased.

Alternatively, the biological micro-objects 1002 that tested positive atstep 108 can be moved from the pens 256 into the channel 252 and flushedby the flow 804 from the channel 252 at step 110. In such an example,the biological micro-objects 1002 that tested positive at both steps 104and 108 can be collected elsewhere in the microfluidic device 200 forstorage, further processing, delivery to another device (not shown), orthe like. The biological micro-objects 1802 that tested negative at step108 can later be removed from the holding pens 256 and discarded.

As shown in FIGS. 28 and 29, the assay material 1910 can be flushed 2802from the channel 434 (FIG. 28). Then, as shown in FIG. 29, thebiological micro-objects 1204, 1206 in the microfluidic device 400 thattested negative at step 108 can be moved from sequestration pens 438,440 into the channel 434 from where the negative biologicalmicro-objects 1204, 1206 can be cleared from the channel 434 (e.g., by aflow of medium (not shown but can be like 2802 of FIG. 28) in thechannel 434). The biological micro-objects 1204, 1206 can be moved fromsequestration pens 438, 440 into the channel 434 in any manner discussedabove (e.g., DEP, gravity, or the like) for moving biologicalmicro-objects 1202, 1204, 1206 from the channel 434 into thesequestration pens 436, 438, 440.

After steps 108 and 110, the process 100 has further sorted themicro-objects (e.g., 1002, 1202, 1204, 1206) selected at step 104 inaccordance with a test performed at step 108. Moreover, themicro-objects selected at step 104 that also tested positive to thesubsequent test at step 108 can remain in the holding pens (e.g., 256,436, 438, 440), while negative micro-objects can be removed.

As discussed above, steps 108 and 110 can be repeated and thus performedn times, where n is an integer one (in which case steps 108 and 110 areperformed once but not repeated) or greater. The subsequent testperformed at each repetition of step 108 can be a different test.Alternatively, the subsequent test performed at a repetition of step 108can be the same test as was previously performed at step 104 or a priorperformance of step 108. The biological micro-objects (e.g., biologicalmicro-objects) loaded at step 102 can thus be subjected to a sequence ofn+1 tests. In some embodiments, each of the n+1 tests can be a differenttest, and in some embodiments, each of the n+1 tests can test for adifferent characteristic. The process 100 can thus sort from initialmixtures of biological micro-objects a group that test positive to n+1tests each of which can be different, and in some embodiments, theprocess 100 can sort from initial mixtures of biological micro-objects agroup that test positive for n+1 different characteristics.

Alternatively, the process 100 can select biological micro-objects atstep 104 and then rank the selected biological micro-objects accordingto the number of tests at step 108 (either performed simultaneously orby repeating step 108) in which the biological micro-objects testpositive. Testing for multiple characteristics in this manner isdesirable for numerous applications, including antibodycharacterization. For example, the multiple tests can help with any ofthe following: identifying conformation specific antibodies (e.g., thedifferent tests can assess the ability of an antibody analyte to binddifferent conformation of a particular antigen); epitope mapping of anantibody analyte (e.g., using genetically or chemically alteredantigen); assessing species cross-reactivity of an antibody analyte(e.g., different tests can assess the ability of antibody analyte tobind to homologous antigens originating from human, mouse, rat, and/orother animals (e.g., experimental animals); and IgG isotyping of anantibody analyte. The generation of chemically modified antigen forepitope mapping of antibodies has been described, for example, inDhungana et al. (2009), Methods Mol. Biol. 524:119-34.

The entire process 100 can be repeated one or more times. Thus, afterperforming steps108 and 110 n times, steps 102-106 can again beperformed k times followed by n more performances of steps 108 and 110.The number k need not be the same number for each repetition of theprocess 100. Similarly, the number n need not be the same number foreach repetition of the process 100. For example, the final repetition ofsteps 108 and 110 for a particular repetition of the process 100, theflow 804 shown in FIG. 27 can load a new mixture of biologicalmicro-objects into the channel 252 of the microfluidic device 200 asillustrated in FIG. 8 and thus be part of step 102 for the nextperformance of the process 100 on the microfluidic device 200.

The process 100 can similarly be repeated multiple times on themicrofluidic device 400. For example, the process 100 can be repeated toretest or reanalyze the positive biological micro-objects kept in theirsequestration pens 436, 438, 440 at step 110; to retest or reanalyzepositive biological micro-objects at reduced density (e.g., onebiological micro-object per sequestration pen, assuming that the initialtest was performed with multiple biological micro-objects persequestration pen); to test or analyze new biological micro-objectsloaded into the microfluidic device 400 at the next repetition of step108; to test or analyze the positive biological micro-objects kept intheir sequestration pens 436, 438, 440 at step 110 with respect to adifferent analyte material (e.g., by repeating step 108 with assaymaterial 1910 designed to detect a second or additional analyte ofinterest); or the like.

FIG. 30 illustrates another example. As shown, after the process 110 hasbeen performed, one or more of the biological micro-objects (e.g., 1202)kept in its sequestration pen (e.g., 436) can be allowed to produce aclonal colony 3002 of biological micro-objects in its sequestration pen(e.g., 436). All or part of the process 100 (e.g., steps 108 and 110)can then be used to test or analyze the colony 3002. Alternatively, thebiological micro-objects can be separated and retested, as discussedabove. In still other alternatives, the biological micro-objects can beallowed to grow into a colony before process 100 has been completed(e.g., after either of steps 106 or 108, but before step 110).

Although specific embodiments and applications of the invention havebeen described in this specification, these embodiments and applicationsare exemplary only, and many variations are possible. For example, theprocess 100 of FIG. 1 and the process 2500 of FIG. 25 are examples only,and variations are contemplated. Thus, for example, at least some of thesteps of process 100 and/or process 2500 can be performed in a differentorder than shown, and some of the steps can be performed simultaneouslyor can otherwise overlap performance of others. As other examples, theprocesses 100, 250 can include additional steps that are not shown orlack some of the steps that are shown.

EXAMPLES Example 1 Screening Mouse Splenocytes for Secretion of IgGantibodies Capable of Binding Human CD45

A screen was performed to identify mouse splenocytes that secreteIgG-type antibodies that bind to human CD45. The experimental designincluded the following steps:

-   -   1. Generation of CD45 antigen coated beads;    -   2. Harvest mouse splenocytes;    -   3. Load cells into a microfluidic device; and    -   4. Assay for antigen specificity.

Reagents used for the experiment included those shown in Table 1.

TABLE 1 Reagents Name Vendor Catalog Number Lot Number 1 Slide-A-LyzerMINI Dialysis Thermo Pierce 69560 OJ189254 Device, 7K MWCO, 0.1 mL 2CD45 Protein R&D Systems 1430-CD 112722 3 PBS pH 7.2 with Mg2+ and Ca2+Fisher BP29404 4 Streptavidin Coated Beads (8 μm) Spherotech SVP-60-5AC01 5 EZ-Fink NHS-PEG4-Biotin, No- Pierce 21329 Weigh Format 6Hybridoma SFM Media Life Tech 12045-076 7 Fetal Bovine Serum Hyclone#SH30084.03 8 Penicillin-Streptomycin Life 15140-122 (10,000 U/mL) 9Goat anti-mouse F(ab′)2-Alexa 568 Life Cat# A11019 Lot#1073003 10streptavidin-488 Life Catalog #S32354 Lot #1078760 11 Mouse anti CD45IgG₁ R&D Systems MAB1430 ILP0612061 12 BD Falcon ™ Cell BD 352340Strainers, 40 μm, Blue

Generation of CD45 Antigen Coated Beads

CD45 antigen coated microbeads were generated in the following manner:

50 μg carrier free CD45 was resuspended in 500 μL PBS (pH 7.2).

A slide-a-lyzer mini cup was rinsed with 500 μL PBS, then added to amicrofuge tube.

50 μL of the 0.1 μg/μL CD45 solution was added to the rinsedslide-a-lyzer mini cup.

170 μL PBS was added to 2 mg of NHS-PEG4-Biotin, after which 4.1 μL ofNHS-PEG4-Biotin was added to the slide-a-lyzer mini cup containing theCD45 antigen.

The NGS-PEG4-Biotin was incubated with the CD45 antigen for 1 hour atroom temperature.

Following the incubation, the slide-a-lyser mini cup was removed fromthe microfuge tube, placed into 1.3 mls PBS (pH 7.2) in a secondmicrofuge tube, and incubated at 4° C. with rocking, for a first 1 hourperiod. The slide-a-lyser mini cup was subsequently transferred to athird microfuge tube containing 1.3 mls of fresh PBS (pH 7.2), andincubated at 4° C. with rocking, for a second 1 hour period. This laststep was repeated three more times, for a total of five 1 hourincubations.

100 μL of biotinylated CD45 solution (˜50 ng/μL) was pipetted intolabeled tubes.

500 μL Spherotech streptavidin coated beads were pipetted into amicrofuge tube, washed 3 times (1000 μL/wash) in PBS (pH 7.4), thencentrifuges for 5 min at 3000 RCF.

The beads were resuspended in 500 μl PBS (pH 7.4), resulting in a beadconcentration of 5 mg/ml.

50 μL biotinylated protein was mixed with the resuspended Spherotechstreptavidin coated beads. The mixture was incubated at 4° C., withrocking, for 2 hours, then centrifuged 4° for 5 min at 3000 RCF. Thesupernatant was discarded and the CD45 coated beads were washed 3 timesin 1 mL PBS (pH 7.4). The beads were then centrifuges at 4° C. foranother 5 min at 3000 RCF. Finally, the CD45 beads were resuspended in500 μL PBS pH 7.4 and stored at 4° C.

Mouse Splenocyte Harvest

The spleen from a mouse immunized with CD45 was harvested and placedinto DMEM media+10% FBS. Scissors were used to mince the spleen.

Minced spleen was placed into a 40 μm cell strainer. Single cells werewashed through the cell strainer with a 10 ml pipette. A glass rod wasused to break up the spleen further and force single cells through thecell strainer, after which single cells were again washed through thecell strainer with a 10 ml pipette.

Red blood cells were lysed with a commercial kit.

Cells were spun down at 200×G and raw splenocytes were resuspended inDMEM media+10% FBS with 10 ml pipette at a concentration of 2e⁸cells/ml.

Loading Cells into Microfluidic Device

Splenocytes were imported into the microfluidic chip and loaded intopens containing 20-30 cells per pen. 100 μL of media was flowed throughthe device at 1 μL/s to remove unwanted cells. Temperature was set to36° C., and culture media was perfused for 30 minutes at 0.1 μL/sec.

Antigen Specificity Assay

Cell media containing 1:2500 goat anti-mouse F(ab′)2-Alexa 568 wasprepared. 100 μL of CD45 beads were re-suspend in 22 μL of the cellmedia containing the 1:2500 dilution of goat anti-mouse F(ab′)2-Alexa568.

The resuspended CD45 beads were next flowed into the main channel of themicrofluidic chip at a rate of 1 μL/sec until they were located adjacentto, but just outside the pens containing splenocytes. Fluid flow wasthen stopped.

The microfluidic chip was then imaged in bright field to determine thelocation of the beads.

Next, a Texas Red Filter was used to capture images of the cells andbeads. Images were taken every 5 minutes for 1 hr, with each exposurelasting 1000 ms and a gain of 5.

Results

Positive signal was observed developing on the beads, reflecting thediffusion of IgG-isotype antibodies diffusing out of certain pens andinto the main channel, where they were able to bind the CD45-coatedbeads. Binding of anti-CD45 antibody to the beads allowed for thesecondary goat anti-mouse IgG-568 to associate with the beads andproduce a detectable signal. See FIGS. 31A-31C & white arrows.

Using the methods of the invention, each group of splenocytes associatedwith positive signal could be separated and moved into new pens as asingle cell and reassayed. In this manner, single cells expressinganti-CD45 IgG antibodies could be detected.

In addition to any previously indicated modification, numerous othervariations and alternative arrangements may be devised by those skilledin the art without departing from the spirit and scope of thisdescription. Thus, while the information has been described above withparticularity and detail in connection with what is presently deemed tobe the most practical and preferred aspects, it will be apparent tothose of ordinary skill in the art that numerous modifications,including, but not limited to, form, function, manner of operation, anduse may be made without departing from the principles and concepts setforth herein. As used herein, the examples and embodiments, in allrespects, are meant to be illustrative only and should not be construedto be limiting in any manner. It should also be noted, that while theterm step is used herein, that term may be used to simply draw attentionto different portions of the described methods and is not meant todelineate a starting point or a stopping point for any portion of themethods, or to be limiting in any other way.

1-28. (canceled)
 29. A microfluidic device, comprising: a cover; asupport structure; and a microfluidic circuit structure disposed betweenthe support structure and the cover; wherein the cover, supportstructure, and microfluidic circuit structure together define amicrofluidic circuit comprising microfluidic circuit components with across-sectional height across the microfluidic circuit, the microfluidiccircuit comprising: a channel configured to contain a flow of a firstfluidic medium, wherein the channel has a uniform cross-sectional heightof about 30 to about 200 microns; and a sequestration pen comprising: anisolation region configured to contain a second fluidic medium; aconnection region containing a proximal opening into the channel, theconnection region fluidically connecting the isolation region with thechannel, wherein the proximal opening between the channel and theconnection region is configured to impede a direct flow of the firstliquid medium into the second liquid medium while allowing diffusionbetween the first fluidic medium and the second fluidic medium.
 30. Themicrofluidic device of claim 29, wherein a width of the channel at theproximal opening of the connection region is in the range of about 50 toabout 1000 microns.
 31. The microfluidic device of claim 30, wherein thewidth of the channel at the proximal opening of the connection region isbetween about 90 microns and about 200 microns.
 32. The microfluidicdevice of claim 29, wherein the cross-sectional height of the channel isabout 30 to about 70 microns.
 33. The microfluidic device of claim 29,wherein a volume of the isolation region ranges from about 1×10⁵ toabout 2×10⁶ cubic meters.
 34. The microfluidic device of claim 29,wherein the microfluidic circuit structure comprises a single substrate.35. The microfluidic device of claim 29, wherein the connection regioncomprises a single proximal opening to the microfluidic channel.
 36. Themicrofluidic device of claim 35, wherein the connection region comprisesa single distal opening to the isolation region.
 37. The microfluidicdevice of claim 29, wherein the proximal opening of the connectionregion is substantially parallel to a direction of the flow of the firstfluidic medium in the channel.
 38. The microfluidic device of claim 29,wherein the proximal opening into the microfluidic channel has a widthW_(con) between about 20 microns to about 100 microns.
 39. Themicrofluidic device of claim 36, wherein a length L_(con) of theconnection region from the proximal opening to the distal opening is atleast about 1.0 times, at least about 1.5 times, or at least about 2.0times the width W_(con) of the proximal opening of the connectionregion.
 40. The microfluidic device of claim 39, wherein the lengthL_(con) of the connection region from the proximal opening to the distalopening and the width W_(con) of the proximal opening of the connectionregion are sized so that a penetration depth into the sequestration penof the first medium flowing in the microfluidic channel at a flow rateno greater than 5.0 uL/sec is less than the length L_(con).
 41. Thedevice of claim 29, wherein the cover and/or the support structure istransparent to light.
 42. The device of claim 29, wherein the cover andthe support structure are part of a dielectrophoresis (DEP) mechanismfor selectively inducing DEP forces on a micro-object.
 43. A method ofanalyzing a biological cell in a microfluidic device, the devicecomprising: a cover; a support structure; and a microfluidic circuitstructure disposed between the support structure and the cover; whereinthe cover, support structure, and microfluidic circuit structuretogether define a microfluidic circuit comprising microfluidic circuitcomponents with a cross-sectional height across the microfluidiccircuit, the microfluidic circuit comprising: a microfluidic channelconfigured to contain a flow of a first fluidic medium, wherein thechannel has a uniform cross-sectional height of about 30 to about 200microns; and a sequestration pen comprising: an isolation regionconfigured to contain a second fluidic medium; a connection regioncontaining a proximal opening into the channel, the connection regionfluidically connecting the isolation region with the microfluidicchannel, wherein the proximal opening between the channel and theconnection region is configured to impede a direct flow of the firstliquid medium into the second liquid medium while allowing diffusionbetween the first fluidic medium and the second fluidic medium. themethod comprising: loading a biological cell into the isolation regionof the sequestration pen; incubating the loaded biological cell for aperiod of time sufficient to allow the biological cell to produce ananalyte of interest; disposing a capture micro-object in themicrofluidic channel at a location in the microfluidic channel adjacentto the proximal opening of the connection region, the capturemicro-object comprising an affinity agent capable of specificallybinding to the analyte of interest; and monitoring binding of thecapture micro-object to the analyte of interest at the location in themicrofluidic channel.
 44. The method of claim 43, wherein disposing thecapture micro-object in the microfluidic channel comprises disposing amixture of capture micro-objects and a labeling agent into themicrofluidic channel.
 45. The method of claim 43, wherein the biologicalcell is selected from the group comprising: B cells, T cells, hybridomacells, cancer cells, embryos and oocytes.
 46. The method of claim 45,wherein loading the biological cell comprises: flowing a group ofbiological cells into the microfluidic channel of the microfluidicdevice; moving a first biological cell of the group into thesequestration pen; and flushing out any biological cells of the groupremaining in the microfluidic channel after loading the first biologicalcell into the sequestration pen.
 47. The method of claim 46, whereinloading the biological cell further comprises selecting the firstbiological cell that meets a predetermined criteria from the group whilethat group is within the microfluidic channel or the connection regionof the sequestration pen.
 48. The method of claim 43, wherein disposingthe capture micro-object further comprises flowing the capturemicro-object in the microfluidic channel, and substantially stopping theflow such that the capture micro-object is adjacent to the proximalopening of the connection region.
 49. The method of claim 43, whereinthe capture micro-object in the microfluidic channel is disposedadjacent to the proximal opening of the connection region to themicrofluidic channel.
 50. The method of claim 43, wherein a lengthL_(con) of the connection region of the sequestration pen is greaterthan a penetration depth D_(p) of the first fluidic medium flowing at amaximum permissible flow rate V_(max) in the microfluidic channel, themethod further comprising keeping any flow of the first fluidic mediumin the microfluidic channel at less than the maximum permissible flowrate V_(max).
 51. The method of claim 43, further comprising flushingthe capture micro-object from the microfluidic channel after monitoringthe binding of the capture micro-object to the analyte of interest. 52.The microfluidic device of claim 29, further comprising a plurality ofchannels configured to contain a flow of the first fluidic medium. 53.The microfluidic device of claim 29, further comprising a plurality ofsequestration pens.
 54. The microfluidic device of claim 39, wherein thelength L_(con) of the connection region is between about 20 microns andabout 500 microns.
 55. The microfluidic device of claim 29, wherein themicrofluidic device further comprises a first electrode, an electrodeactivation substrate, and a second electrode, wherein the firstelectrode is part of a first wall of an enclosure and the electrodeactivation substrate and the second electrode is part of a second wallof the enclosure, wherein the electrode activation substrate comprises aphotoconductive material, semiconductor integrated circuits, orphototransistors.
 56. The method of claim 43, wherein the labeling agentcomprises a fluorescent label.